Sc-beta cells and compositions and methods for generating the same

ABSTRACT

Disclosed herein are methods, compositions, kits, and agents useful for inducing β cell maturation, and isolated populations of SC-β cells for use in various applications, such as cell therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 14/684,129, filed on Apr. 10, 2015, which is a continuation of PCTApplication No. PCT/US2014/041992, filed Jun. 11, 2014, which claims thebenefit of U.S. Provisional Application No. 61/833,898, filed on Jun.11, 2013, and U.S. Provisional Application No. 61/972,212, filed on Mar.28, 2014, the contents of all of which are hereby incorporated byreference in their entirety.

BACKGROUND OF THE INVENTION

Research to-date has generated only abnormally functioninginsulin-expressing cells, which do not secrete appropriate amounts ofinsulin in response to sequentially varied glucose levels, or pancreaticprogenitor cells that can only mature into functioninginsulin-expressing cells after 3 months of transplantation into a mousehost (Cheng et. al., 2012; D'Amour et al., 2005; D'Amour et al., 2006;Kroon et al., 2008; Nostro et al., 2011; Rezania et al., 2012; Schulz etal., 2012; Xie et al., 2013). In contrast to normal islets or dispersedadult β cells, which release high levels of insulin in response to highlevels of glucose in the “glucose stimulated insulin secretion” (GSIS)assay and can do so repeatedly, hPSC-derived insulin-expressing cellsgenerated by existing methods fail to secrete insulin appropriately inresponse to the addition of various concentrations of glucose.Accordingly, there exists a need for a method of deriving cells fromhPSCs which exhibit a phenotype of normal islets or mature adult βcells.

SUMMARY OF THE INVENTION

In some aspects, the disclosure provides a stem cell-derived β cell(SC-β).

In some embodiments, the cell is mature. In some embodiments, the cellexhibits an in vitro glucose stimulated insulin secretion (GSIS)response. In some embodiments, the cell exhibits an iv vivo GSISresponse. In some embodiments, the cell exhibits in vitro and in vivoglucose stimulated insulin secretion (GSIS) responses. In someembodiments, the cell exhibits a GSIS response to at least one glucosechallenge. In some embodiments, the cell exhibits a GSIS response to atleast two sequential glucose challenges. In some embodiments, the cellexhibits a GSIS response to at least three sequential glucosechallenges. In some embodiments, the GSIS response is observedimmediately upon transplanting the cell into a human or animal. In someembodiments, the GSIS response is observed within approximately 24 hoursof transplanting the cell into a human or animal. In some embodiments,the GSIS response is observed within approximately two weeks oftransplanting the cell into a human or animal. In some embodiments, thestimulation index of the cell as characterized by the ratio of insulinsecreted in response to high glucose concentrations compared to lowglucose concentrations is similar to the stimulation index of anendogenous mature pancreatic β cell. In some embodiments, thestimulation index is greater than or equal to 1, or greater than orequal to 1.1, or greater than or equal to 1.3, or greater than or equalto 2, or greater than or equal to 2.3, or greater than or equal to 2.6.In some embodiments, the cell exhibits cytokine-induced apoptosis inresponse to a cytokine. In some embodiments, the cytokine is selectedfrom the group consisting of interleukin-1β (IL-β), interferon-γ(INF-γ), tumor necrosis factor-α (TNF-α), and combinations thereof. Insome embodiments, insulin secretion from the cell is enhanced inresponse to an anti-diabetic agent. In some embodiments, theanti-diabetic agent comprises a secretagogue selected from the groupconsisting of an incretin mimetic, a sulfonylurea, a meglitinide, andcombinations thereof. In some embodiments, the cell is monohormonal. Insome embodiments, the cell exhibits a morphology that resembles themorphology of an endogenous mature pancreatic β cell. In someembodiments, the cell exhibits encapsulated crystalline insulin granulesunder electron microscopy that resemble insulin granules of anendogenous mature pancreatic β cell. In some embodiments, the cellexhibits a low rate of replication. In some embodiments, the cellexhibits a glucose stimulated Ca²⁺ flux (GSCF) that resembles the GSCFof an endogenous mature pancreatic β cell. In some embodiments, the cellexhibits a GSCF response to at least one glucose challenge. In someembodiments, the cell exhibits a GSCF response to at least two glucosechallenges. In some embodiments, the cell exhibits a GSCF response to atleast three glucose challenges. In some embodiments, the cell exhibitsan increased calcium flux. In some embodiments, the increased calciumflux comprises an increased amount of influx or a ratio of influx at lowrelative to high glucose concentrations. In some embodiments, the cellexpresses at least one marker characteristic of an endogenous maturepancreatic β cell selected from the group consisting of insulin,C-peptide, PDX1, MAFA, NKX6-1, PAX6, NEUROD1, glucokinase (GCK), SLC2A1,PCSK1, KCNJ11, ABCC8, SLC30A8, SNAP25, RAB3A, GAD2, PTPRN, NKX2-2, Pax4.In some embodiments, the cell does not express at least one markerselected from the group consisting of a) a hormone selected from thegroup consisting of i) glucagon (GCG), and ii) somatostatin (SST); or b)an acinar cell marker selected from the group consisting of i) amylase,and ii) carboxypeptdase A (CPA1); c) an a cell marker selected from thegroup consisting of i) GCG, ii) Arx, iii) Irx1, and Irx2; and d) aductal cell marker selected from the group consisting of i) CFTR, andii) Sox9. In some embodiments, the cell is differentiated in vitro froman insulin-positive endocrine cell or a precursor thereof selected fromthe group consisting of a Nkx6-1-positive pancreatic progenitor cell, aPdx1-positive pancreatic progenitor cell, and a pluripotent stem cell.In some embodiments, the pluripotent stem cell is selected from thegroup consisting of an embryonic stem cell and induced pluripotent stemcell. In some embodiments, the cell is human. In some embodiments, thecell is not genetically modified. In some embodiments, the cell isgenetically modified. In some embodiments, the insulin produced per cellis between 0.5 and 10 μIU per 1000 cells per 30 minute incubation at ahigh glucose concentration. In some embodiments, the insulin producedper cell is approximately 2.5 μIU per 1000 cells per 30 minuteincubation at a high glucose concentration. In some embodiments, theincubation occurs ex vivo.

In some aspects, the disclosure provides a cell line comprising a SC-βcell. In some embodiments, the cell line stably expresses insulin. Insome embodiments, the cells can be frozen, thawed, and amplified with adoubling time of between about 24 and 44 hours without significantmorphological changes until at least 30 passages.

In some aspects, the disclosure provides a method of generating a SC-βcell from insulin-positive endocrine cells, the method comprisingcontacting a population of cells comprising insulin-positive endocrinecells under conditions that promote cell clustering with at least two βcell-maturation factors comprising a) a transforming growth factor β(TGF-β) signaling pathway inhibitor and b) a thyroid hormone signalingpathway activator, to induce the in vitro maturation of at least oneinsulin-positive endocrine cell in the population into a SC-β cell.

In some embodiments, the SC-β cell exhibits a response to at least oneglucose challenge. In some embodiments, the SC-β cell exhibits aresponse to at least two sequential glucose challenges. In someembodiments, the SC-β cell exhibits a response to at least threesequential glucose challenges. In some embodiments, the morphology ofthe SC-β cell resembles the morphology of an endogenous mature β cell.In some embodiments, the SC-β cell exhibits in vitro and/or in vivoglucose stimulated insulin secretion (GSIS) responses. In someembodiments, the GSIS response is observed immediately upontransplantation of the SC-β cell into a subject. In some embodiments,the GSIS response is observed within approximately 24 hours upontransplantation of the SC-β cell into a subject. In some embodiments,the GSIS response is observed within approximately two weeks oftransplantation of the SC-β cell into a subject. In some embodiments,the population of cells is contacted with the TGF-β signaling pathwayinhibitor at a concentration of between 100 nM-100 μM. In someembodiments, the population of cells is contacted with the TGF-βsignaling pathway inhibitor at a concentration of 10 μM. In someembodiments, the TGF-β signaling pathway comprises TGF-β receptor type Ikinase signaling. In some embodiments, the TGF-β signaling pathwayinhibitor comprises Alk5 inhibitor II. In some embodiments, the TGF-βsignaling pathway inhibitor comprises an analog or derivative of Alk5inhibitor II. In some embodiments, the population of cells is contactedwith the thyroid hormone signaling pathway activator at a concentrationof between 0.1 μM-10 μM. In some embodiments, the population of cells iscontacted with the thyroid hormone signaling pathway activator at aconcentration of 1 μM. In some embodiments, the thyroid hormonesignaling pathway activator comprises triiodothyronine (T3). In someembodiments, the population of cells is optionally contacted with aprotein kinase inhibitor. In some embodiments, the population of cellsis not contacted with the protein kinase inhibitor. In some embodiments,the population of cells is contacted with the protein kinase inhibitor.In some embodiments, the population of cells is contacted with theprotein kinase inhibitor at a concentration of between 10 nM-1 μM. Insome embodiments, the population of cells is contacted with the proteinkinase inhibitor at a concentration of 100 nM. In some embodiments, theprotein kinase inhibitor comprises staurosporine. In some embodiments,the method includes contacting the population of cells with at least oneadditional β cell-maturation factor. In some embodiments, the at leastone additional β cell-maturation factor comprises a cystic fibrosistransmembrane conductance regulator (CFTR) inhibitor. In someembodiments, the population of cells is contacted with the CFTRinhibitor at a concentration of between 100 nM-100 μM. In someembodiments, the population of cells is contacted with the CFTRinhibitor at a concentration of between 10 nM-10 μM. In someembodiments, the CFTR inhibitor comprises Gly-H101. In some embodiments,the at least one additional β cell-maturation factor comprises aO-GlcNAcase inhibitor. In some embodiments, the population of cells iscontacted with the O-GlcNAcase inhibitor at a concentration of between100 nM-100 μM. In some embodiments, the population of cells is contactedwith the O-GlcNAcase inhibitor at a concentration of 10 nM-10 μM. Insome embodiments, the inhibitor of O-GlcNAcase comprises Thiamet G. Insome embodiments, the population of cells is cultured in a suitableculture medium. In some embodiments, the suitable culture mediumcomprises Connought Medical Research Laboratories 1066 supplementedislet media (CMRLS) or a component of CMRLS. In some embodiments, theCMRLS is supplemented with serum. In some embodiments, the CMRLS issupplemented with 10% fetal bovine serum. In some embodiments, theconditions that promote cell clustering comprise a suspension culture.In some embodiments, the population of cells is maintained in asuspension culture for a period of time sufficient to induce the invitro maturation of at least one of the insulin-positive endocrine cellsin the population of cells into at least one SC-β cell. In someembodiments, the period of time comprises at least 7 days. In someembodiments, the period of time comprises between 7 days and 21 days. Insome embodiments, the period of time comprises between 7 and 14 days. Insome embodiments, the period of time comprises between 10 and 14 days.In some embodiments, the period of time comprises 14 days. In someembodiments, the β cell-maturation factors are replenished every otherday. In some embodiments, at least 1% of the insulin-positive endocrinecells in the population of cells are induced to mature into SC-β cells.In some embodiments, at least 99% of the insulin-positive endocrinecells in the population are induced to mature into SC-β cells. In someembodiments, at least 30% of the resulting cells in the populationcomprise SC-β cells. In some embodiments, the SC-β cells expressC-peptide, insulin, NKX6-1, Pdx1, and co-express NKX6-1 and C-peptide.In some embodiments, the insulin-positive endocrine cells also expressPdx1 and NKX6-1. In some embodiments, the insulin-positive endocrinecells are produced from a population of pluripotent stem cells selectedfrom the group consisting of embryonic stem cells and inducedpluripotent stem cells. In some embodiments, the SC-β cells comprisehuman cells. In some embodiments, the generation of SC-β cells in vitrois scalable.

In some aspects, the disclosure provides an isolated population of SC-βcells produced according to the methods described herein.

In some aspects, the disclosure provides a microcapsule comprising anisolated population of SC-β cells encapsulated therein.

In some aspects, the disclosure provides a composition comprising apopulation of SC-β cells produced according to a method describedherein.

In some aspects, the disclosure provides an assay comprising an isolatedpopulation of SC-β cells produced according to a method describedherein.

In some embodiments, the assay is for use in identifying one or morecandidate agents which promote or inhibit a β cell fate selected fromthe group consisting of β cell proliferation, β cell replication, β celldeath, β cell function, β cell susceptibility to immune attack, or βcell susceptibility to dedifferentiation or differentiation. In someembodiments, the assay is for use in identifying one or more candidateagents which promote the differentiation of at least oneinsulin-positive endocrine cell or a precursor thereof into at least oneSC-β cell.

In some aspects, the disclosure provides a method for the treatment of asubject in need thereof, the method comprising administering to asubject a composition comprising an isolated population of SC-β cellsproduced according a method described herein. In some embodiments, theSC-β cells are encapsulated in a microcapsule. In some embodiments, theSC-β cells are produced from a population of pluripotent stem cellsobtained from the same subject that the SC-β cells are administered to.In some embodiments, the SC-β cells are produced from a population ofiPS cells, wherein the iPS cells are derived from a cell obtained fromthe same subject that the SC-β cells are administered to. In someembodiments, the subject has, or has an increased risk of developing,diabetes. In some embodiments, the diabetes is selected from the groupof Type I diabetes, Type II diabetes, Type 1.5 diabetes andpre-diabetes. In some embodiments, the subject has, or has an increasedrisk of developing a metabolic disorder.

In some aspects, the disclosure relates to the use of an isolatedpopulation of SC-β cells produced by the methods described herein foradministering to a subject in need thereof.

In some embodiments, the isolated population of SC-β cells isadministered to the subject encapsulated in microcapsules. In someembodiments, the subject has, or has an increased risk of developingdiabetes. In some embodiments, the diabetes is selected from the groupof Type I diabetes, Type II diabetes, Type 1.5 diabetes andpre-diabetes. In some embodiments, the subject has, or has an increasedrisk of developing a metabolic disorder.

In some aspects, the disclosure provides a culture medium comprising a)Alk5 inhibitor, b) triiodothyronine (T3), optionally c) staurosporine,and optionally d) CMRLS or a component of CMRLS.

In some aspects, the disclosure involves the use of the culture mediumof to induce the in vitro maturation of insulin-positive endocrine cellsinto SC-β cells, wherein the SC-β cells exhibit both an in vitro and/orin vivo GSIS response.

In some aspects, the disclosure provides a method of producing aNKX6-1-positive pancreatic progenitor cell from a Pdx1-positivepancreatic progenitor cell comprising contacting a population of cellscomprising Pdx1-positive pancreatic progenitor cells under conditionsthat promote cell clustering with at least two β cell-maturation factorscomprising a) at least one growth factor from the fibroblast growthfactor (FGF) family, b) a sonic hedgehog pathway inhibitor, andoptionally c) a low concentration of a retinoic acid (RA) signalingpathway activator, for a period of at least five days to induce thedifferentiation of at least one Pdx1-positive pancreatic progenitor cellin the population into NKX6-1-positive pancreatic progenitor cells,wherein the NKX6-1-positive pancreatic progenitor cells express NKX6-1.

In some embodiments, the population of cells is contacted with the atleast one growth factor from the FGF family at a concentration ofbetween 1 ng/mL-100 ng/mL. In some embodiments, the population of cellsis contacted with the at least one growth factor from the FGF family ata concentration of 50 ng/mL. In some embodiments, the at least onegrowth factor from the FGF family comprises keratinocyte growth factor(KGF). In some embodiments, the at least one growth factor from the FGFfamily is selected from the group consisting of FGF2, FGF8B, FGF10, andFGF21. In some embodiments, the population of cells is not contactedwith the RA signaling pathway activator. In some embodiments, thepopulation of cells is contacted with the RA signaling pathway activatorat a concentration of between 0.01 μM-1.0 μM. In some embodiments, thepopulation of cells is contacted with the RA signaling pathway activatorat a concentration of 0.1 μM. In some embodiments, the RA signalingpathway activator comprises RA. In some embodiments, the population ofcells is contacted with the SHH pathway inhibitor at a concentration ofbetween 0.1 μM and 0.5 μM. In some embodiments, the population of cellsis contacted with the SHH pathway inhibitor at a concentration of 0.25μM. In some embodiments, the SHH pathway inhibitor comprises Sant1. Insome embodiments, the method includes exposing the population of cellsto at least one additional β cell-maturation factor. In someembodiments, the at least one additional β cell-maturation factorcomprises at least one growth factor from the EGF family. In someembodiments, the population of cells is exposed to the at least onegrowth factor from the EGF family at a concentration of between 2ng/mL-200 ng/mL. In some embodiments, the population of cells is exposedto the at least one growth factor from the EGF family at a concentrationof 20 ng/mL. In some embodiments, at least one growth factor from theEGF family is selected from the group consisting of betacellulin andEGF. In some embodiments, the population of cells is cultured in asuitable culture medium. In some embodiments, the conditions thatpromote cell clustering comprise a suspension culture. In someembodiments, the β cell-maturation factors are replenished every otherday. In some embodiments, an activator of protein kinase C is not addedto the suspension culture during the 5 days. In some embodiments, anactivator of protein kinase C is removed from the suspension cultureprior to the 5 days. In some embodiments, the activator of proteinkinase C comprises PdbU. In some embodiments, a BMP signaling pathwayinhibitor is not added to the suspension culture during the 5 days. Insome embodiments, a BMP signaling pathway inhibitor is removed from thesuspension culture prior to the 5 days. In some embodiments, the BMPsignaling pathway inhibitor comprises LDN193189. In some embodiments, atleast 10% of the Pdx1-positive pancreatic progenitor cells in thepopulation are induced to differentiate into NKX6-1-positive pancreaticprogenitor cells. In some embodiments, at least 95% of the Pdx1-positivepancreatic progenitor cells in the population are induced todifferentiate into NKX6-1-positive pancreatic progenitor cells. In someembodiments, the NKX6-1-positive pancreatic progenitor cells expressPdx1, NKX6-1, and FoxA2. In some embodiments, the Pdx1-positivepancreatic progenitor cells are produced from a population ofpluripotent stem cells selected from the group consisting of embryonicstem cells and induced pluripotent stem cells.

In some aspects, the disclosure provides an isolated population ofNKX6-1-positive pancreatic progenitor cells obtained by a methoddescribed herein.

In some aspects, the disclosure provides a microcapsule comprising theisolated population of NKX6-1-positive pancreatic progenitor cellsencapsulated therein.

In some aspects, the disclosure provides a composition comprising anisolated population of NKX6-1-positive pancreatic progenitor cellsproduced according to a method described herein.

In some aspects, the disclosure provides an assay comprising an isolatedpopulation of NKX6-1-positive pancreatic progenitor cells producedaccording to a method described herein.

In some embodiments, the assay is for use in identifying one or morecandidate agents which promote the differentiation of at least onePdx1-positive pancreatic progenitor cell or precursor thereof intoNKX6-1-positive pancreatic progenitor cells.

In some aspects, the disclosure provides a method for the treatment of asubject in need thereof, the method comprising administering to asubject a composition comprising an isolated population ofNKX6-1-positive pancreatic progenitor cells produced according to amethod described herein.

In some embodiments, the NKX6-1-positive pancreatic progenitor cells areproduced from a population of pluripotent stem cells obtained from thesame subject as the NKX6-1-positive pancreatic progenitor cells areadministered to. In some embodiments, the NKX6-1-positive pancreaticprogenitor cells are encapsulated in a microcapsule. In someembodiments, the subject has, or has an increased risk of developingdiabetes. In some embodiments, the diabetes is selected from the groupof Type I diabetes, Type II diabetes, Type 1.5 diabetes andpre-diabetes. In some embodiments, the subject has, or has an increasedrisk of developing a metabolic disorder.

In some aspects, the disclosure relates to the use of an isolatedpopulation of NKX6-1-positive pancreatic progenitor cells produced bythe methods described herein for differentiating into SC-β cells.

In some aspects, the disclosure involves the use of an isolatedpopulation of NKX6-1-positive pancreatic progenitor cells produced by amethod described herein for administering to a subject in need thereof.

In some embodiments, the isolated population of NKX6-1-positivepancreatic progenitor cells is administered to the subject encapsulatedin microcapsules. In some embodiments, the subject has, or has anincreased risk of developing diabetes. In some embodiments, the diabetesis selected from the group of Type I diabetes, Type II diabetes, Type1.5 diabetes and pre-diabetes. In some embodiments, the subject has, orhas an increased risk of developing a metabolic disorder.

In some aspects, the disclosure provides a culture medium comprising a)KGF, b) SANT1), and optionally c) RA, wherein the culture medium issubstantially free of PdbU and LDN 193189. In some embodiments, thedisclosure involves the use of the culture medium described herein toinduce the in vitro differentiation of Pdx1-positive pancreaticprogenitor cells into NKX6-1-positive pancreatic progenitor cells.

In some aspects, the disclosure provides a method of producing aninsulin-positive endocrine cell from an NKX6-1-positive pancreaticprogenitor cell comprising contacting a population of cells comprisingNKX6-1-positive pancreatic progenitor cells under conditions thatpromote cell clustering with at least two β cell-maturation factorscomprising a) a TGF-β signaling pathway inhibitor, and b) thyroidhormone signaling pathway activator, to induce the differentiation of atleast one NKX6-1-positive pancreatic progenitor cell in the populationinto at least one insulin-positive endocrine cell, wherein theinsulin-positive pancreatic progenitor cell expresses insulin. In someembodiments, the population of cells is contacted with the TGF-βsignaling pathway inhibitor at a concentration of between 100 nM-100 μM.In some embodiments, the population of cells is contacted with the TGF-βsignaling pathway inhibitor at a concentration of 10 μM. In someembodiments, the TGF-β signaling pathway comprises TGF-β receptor type Ikinase signaling. In some embodiments, the TGF-β signaling pathwayinhibitor comprises Alk5 inhibitor II. In some embodiments, thepopulation of cells is contacted with the thyroid hormone signalingpathway activator at a concentration of between 0.1 μM-10 μM. In someembodiments, the population of cells is contacted with the thyroidhormone signaling pathway activator at a concentration of 1 μM. In someembodiments, the thyroid hormone signaling pathway activator comprisestriiodothyronine (T3). In some embodiments, the method includescontacting the population of cells with at least one additional βcell-maturation factor. In some embodiments, the at least one additionalβ cell-maturation factor comprises a γ-secretase inhibitor. In someembodiments, the population of cells is contacted with the γ-secretaseinhibitor at a concentration of between 0.1 μM-10 μM. In someembodiments, the population of cells is contacted with the γ-secretaseinhibitor at a concentration of 1 μM. In some embodiments, theγ-secretase inhibitor comprises XXI. In some embodiments, theγ-secretase inhibitor comprises DAPT. In some embodiments, the at leastone additional β cell-maturation factor comprises at least one growthfactor from the EGF family. In some embodiments, the population of cellsis contacted with the at least one growth factor from the EGF family ata concentration of between 2 ng/mL-200 ng/mL. In some embodiments, thepopulation of cells is contacted with at least one growth factor fromthe EGF family at a concentration of 20 ng/mL. In some embodiments, theat least one growth factor from the EGF family comprises betacellulin.In some embodiments, the at least one growth factor from the EGF familycomprises EGF. In some embodiments, the at least one additional βcell-maturation factor comprises a low concentration of a retinoic acid(RA) signaling pathway activator. In some embodiments, the population ofcells is contacted with the RA signaling pathway activator at aconcentration of between 0.01 μM-1.0 μM. In some embodiments, thepopulation of cells is contacted with the RA signaling pathway activatorat a concentration of 0.1 μM. In some embodiments, the RA signalingpathway activator comprises RA. In some embodiments, the at least oneadditional β cell-maturation factor comprises a sonic hedgehog (SHH)pathway inhibitor. In some embodiments, the population of cells iscontacted with the SHH pathway inhibitor at a concentration of between0.1 μM and 0.5 μM. In some embodiments, the population of cells iscontacted with the SHH pathway inhibitor at a concentration of 0.25 μM.In some embodiments, the SHH pathway inhibitor comprises Sant1. In someembodiments, the population of cells is optionally contacted with aprotein kinase inhibitor. In some embodiments, the population of cellsis not contacted with the protein kinase inhibitor. In some embodiments,the population of cells is contacted with the protein kinase inhibitor.In some embodiments, the population of cells is contacted with theprotein kinase inhibitor at a concentration of between 10 nM-1 μM. Insome embodiments, the population of cells is contacted with the proteinkinase inhibitor at a concentration of 100 nM. In some embodiments, theprotein kinase inhibitor comprises staurosporine. In some embodiments,the method includes exposing the population of cells to glucose. In someembodiments, the population of cells is exposed to glucose at aconcentration of between 1 mM-50 mM. In some embodiments, the populationof cells is exposed to glucose at a concentration of 25 mM. In someembodiments, the conditions that promote cell clustering comprise asuspension culture. In some embodiments, the population of cells ismaintained in suspension culture for a period of time sufficient toinduce the differentiation of at least one of the NKX6-1-positivepancreatic progenitor cells in the population into an insulin-positiveendocrine cell. In some embodiments, the period of time is at least 7days. In some embodiments, the β cell-maturation factors are replenishedin the suspension culture every other day. In some embodiments, at least15% of the NKX6-1-positive pancreatic progenitor cells in the populationare induced to differentiate into insulin-positive endocrine cells. Insome embodiments, at least 99% of the NKX6-1-positive pancreaticprogenitor cells in the population are induced to differentiate intoinsulin-positive endocrine cells. In some embodiments, theinsulin-positive endocrine cells express Pdx1, NKX6-1, NKX2-2, Mafb,glis3, Sur, Kir6.2, Znt8, SLC2A1, SLC2A3 and/or insulin. In someembodiments, the NKX6-1-positive pancreatic progenitor cells areproduced from a population of pluripotent stem cells selected from thegroup consisting of embryonic stem cells and induced pluripotent stemcells.

In some aspects, the disclosure provides an isolated population ofinsulin-positive endocrine cells produced according to a methoddescribed herein.

In some aspects, the disclosure provides a microcapsule comprising theisolated population of insulin-positive endocrine cells encapsulatedtherein. In some embodiments, the disclosure provides a compositioncomprising a population of insulin-positive endocrine cells producedaccording to a method described herein.

In some aspects, the disclosure provides a method for the treatment of asubject in need thereof, the method comprising administering to asubject a composition comprising an isolated population ofinsulin-positive endocrine cells produced according to a methoddescribed herein.

In some embodiments, the insulin-positive endocrine cells are producedfrom a population of pluripotent stem cells obtained from the samesubject as the insulin-positive endocrine cells are administered to. Insome embodiments, the insulin-positive endocrine cells are encapsulatedin a microcapsule. In some embodiments, the subject has, or has anincreased risk of developing diabetes. In some embodiments, the diabetesis selected from the group of Type I diabetes, Type II diabetes, Type1.5 diabetes and pre-diabetes. In some embodiments, the subject has, orhas an increased risk of developing a metabolic disorder.

In some aspects, the disclosure involves the use of an isolatedpopulation of insulin-positive endocrine cells produced by a methoddescribed herein for differentiating into SC-β cells.

In some aspects, the disclosure involves the use of an isolatedpopulation of insulin-positive endocrine cells produced by a methoddescribed herein for administering to a subject in need thereof.

In some embodiments, the isolated population of insulin-positiveendocrine cells is administered to the subject encapsulated inmicrocapsules. In some embodiments, the subject has, or has an increasedrisk of developing diabetes. In some embodiments, the diabetes isselected from the group of Type I diabetes, Type II diabetes, Type 1.5diabetes and pre-diabetes. In some embodiments, the subject has, or hasan increased risk of developing a metabolic disorder.

In some aspects, the disclosure provides a culture medium comprising a)TGF-β signaling pathway inhibitor, b) a TH pathway activator, and atleast one additional β cell-maturation factor selected from the groupconsisting of i) XXI, ii) Betacellulin, iii) a low concentration of a RAsignaling pathway activator, and iv) a SHH pathway inhibitor.

In some aspects, the disclosure involves the use of the culture mediumdescribed herein to induce the in vitro differentiation ofNKX6-1-positive pancreatic progenitor cells into insulin-positiveendocrine cells.

In some aspects, the disclosure provides a method of generating SC-βcells, the method comprising: contacting Pdx1-positive, NKX6-1-positive,insulin-positive endocrine cells under conditions that promote cellclustering with i) a transforming growth factor β (TGF-β) signalingpathway inhibitor, ii) a thyroid hormone signaling pathway activator,and optionally iii) a protein kinase inhibitor, to induce the in vitromaturation of at least some of the Pdx1-positive, NKX6-1-positive,insulin-positive endocrine cells into SC-β cells, wherein the SC-β cellsexhibit a GSIS response in vitro and/or in vivo.

In some embodiments, the GSIS response is observed (i) immediately upontransplantation of the SC-β cell into a subject; (ii) withinapproximately 24 hours of transplantation into a subject; or (iii)within approximately two weeks of transplantation into a subject. Insome embodiments, the SC-β cells exhibit a response to (i) at least oneglucose challenge; (ii) at least two sequential glucose challenges; or(iii) at least three sequential glucose challenges. In some embodiments,the morphology of the SC-β cells resembles the morphology of endogenousβ cells. In some embodiments, the Pdx1-positive, NKX6-1-positive,insulin-positive endocrine cells are contacted with the TGF-β signalingpathway inhibitor at a concentration of between 100 nM-100 μM. In someembodiments, the Pdx1-positive, NKX6-1-positive, insulin-positiveendocrine cells are contacted with the TGF-β signaling pathway inhibitorat a concentration of 10 μM. In some embodiments, the TGF-β signalingpathway comprises TGF-β receptor type I kinase signaling. In someembodiments, the TGF-β signaling pathway inhibitor comprises Alk5inhibitor II. In some embodiments, the Pdx1-positive, NKX6-1-positive,insulin-positive endocrine cells are contacted with the thyroid hormonesignaling pathway activator at a concentration of between 0.1 μM-10 μM.In some embodiments, the Pdx1-positive, NKX6-1-positive,insulin-positive endocrine cells are contacted with the thyroid hormonesignaling pathway activator at a concentration of 1 μM. In someembodiments, the thyroid hormone signaling pathway activator comprisestriiodothyronine (T3). In some embodiments, the Pdx1-positive,NKX6-1-positive, insulin-positive endocrine cells are not contacted withthe protein kinase inhibitor. In some embodiments, the Pdx1-positive,NKX6-1-positive, insulin-positive endocrine cells are contacted with theprotein kinase inhibitor. In some embodiments, the Pdx1-positive,NKX6-1-positive, insulin-positive endocrine cells are contacted with theprotein kinase inhibitor at a concentration of between 10 nM-1 μM. Insome embodiments, the Pdx1-positive, NKX6-1-positive, insulin-positiveendocrine cells are contacted with the protein kinase inhibitor at aconcentration of 100 nM. In some embodiments, the protein kinaseinhibitor comprises staurosporine. In some embodiments, the methodincludes contacting the Pdx1-positive, NKX6-1-positive, insulin-positiveendocrine cells with a cystic fibrosis transmembrane conductanceregulator (CFTR) inhibitor. In some embodiments, the Pdx1-positive,NKX6-1-positive, insulin-positive endocrine cells are contacted with theCFTR inhibitor at a concentration of between 100 nM and 100 μM. In someembodiments, the Pdx1-positive, NKX6-1-positive, insulin-positiveendocrine cells are contacted with the CFTR inhibitor at a concentrationof 10 nM and 10 uM. In some embodiments, the CFTR inhibitor comprisesGly-H101. In some embodiments, the method includes contacting thePdx1-positive, NKX6-1-positive, insulin-positive endocrine cells with aO-GlcNAcase inhibitor. In some embodiments, the Pdx1-positive,NKX6-1-positive, insulin-positive endocrine cells are contacted with the0-GlcNAcase inhibitor at a concentration of between 100 nM and 100 μM.In some embodiments, the Pdx1-positive, NKX6-1-positive,insulin-positive endocrine cells are contacted with the O-GlcNAcaseinhibitor at a concentration of between 10 nM and 10 uM. In someembodiments, the inhibitor of O-GlcNAcase comprises Thiamet G. In someembodiments, the Pdx1-positive, NKX6-1-positive, insulin-positiveendocrine cells are cultured in a suitable culture medium. In someembodiments, the suitable culture medium comprises Connought MedicalResearch Laboratories 1066 supplemented islet media (CMRLS) or acomponent of CMRLS. In some embodiments, the CMRLS is supplemented withserum. In some embodiments, the CMRLS is supplemented with 10% fetalbovine serum. In some embodiments, the conditions that promote cellclustering comprise suspension culture. In some embodiments, thePdx1-positive, NKX6-1-positive, insulin-positive endocrine cells aremaintained in a suspension culture for a period of time sufficient toinduce the in vitro maturation of at least some of the Pdx1-positive,NKX6-1-positive, insulin-positive endocrine cells into SC-β cells. Insome embodiments, the period of time comprises at least 7 days. In someembodiments, the period of time comprises between 7 days and 21 days. Insome embodiments, the period of time comprises between 7 and 14 days. Insome embodiments, the period of time comprises 14 days. In someembodiments, the suspension culture is replenished every other day. Insome embodiments, at least 30% of the cells generated comprise SC-βcells. In some embodiments, the SC-β cells express C-peptide, insulin,NKX6-1, Pdx1, and co-express NKX6-1 and C-peptide. In some embodiments,the SC-β cells comprise human cells. In some embodiments, the generationof the SC-β cells in vitro is scalable.

In some embodiments, the insulin-positive, endocrine cells are obtainedby contacting Pdx1-positive, NKX6-1-positive pancreatic progenitor cellsunder conditions that promote cell clustering with i) a TGF-β signalingpathway inhibitor, and ii) a thyroid hormone signaling pathwayactivator, to induce the differentiation of at least some of thePdx1-positive, NKX6-1-positive pancreatic progenitor cells intoPdx1-positive, NKX6-1-positive, insulin-positive endocrine cells,wherein the Pdx1-positive, NKX6-1-positive, insulin-positive endocrinecells express Pdx1, NKX6-1, NKX2-2, Mafb, glis3, Sur, Kir6.2, Znt8,SLC2A1, SLC2A3 and/or insulin.

In some embodiments, the Pdx1-positive, NKX6-1-positive pancreaticprogenitor cells are contacted with the TGF-β signaling pathwayinhibitor at a concentration of between 100 nM-100 μM. In someembodiments, the Pdx1-positive, NKX6-1-positive pancreatic progenitorcells are contacted with the TGF-β signaling pathway inhibitor at aconcentration of 10 μM. In some embodiments, the TGF-β signaling pathwaycomprises TGF-β receptor type I kinase signaling. In some embodiments,the TGF-β signaling pathway inhibitor comprises Alk5 inhibitor II. Insome embodiments, the Pdx1-positive, NKX6-1-positive pancreaticprogenitor cells are contacted with the thyroid hormone signalingpathway activator at a concentration of between 0.1 μM-10 μM. In someembodiments, the Pdx1-positive, NKX6-1-positive pancreatic progenitorcells are contacted with the thyroid hormone signaling pathway activatorat a concentration of 1 μM. In some embodiments, the thyroid hormonesignaling pathway activator comprises triiodothyronine (T3). In someembodiments, the method includes contacting the Pdx1-positiveNKX6-1-positive pancreatic progenitor cells with at least one of i) aSHH pathway inhibitor, ii) a RA signaling pathway activator, iii) aγ-secretase inhibitor, iv) at least one growth factor from the epidermalgrowth factor (EGF) family, and optionally v) a protein kinaseinhibitor. In some embodiments, the Pdx1-positive, NKX6-1-positivepancreatic progenitor cells are contacted with the SHH pathway inhibitorat a concentration of between 0.1 μM and 0.5 μM. In some embodiments,the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells arecontacted with a SHH pathway inhibitor at a concentration of 0.25 μM. Insome embodiments, the SHH pathway inhibitor comprises Sant1. In someembodiments, the Pdx1-positive, NKX6-1-positive pancreatic progenitorcells are contacted with the RA signaling pathway activator at aconcentration of between 0.01 μM-1.0 μM. In some embodiments, thePdx1-positive, NKX6-1-positive pancreatic progenitor cells are contactedwith the RA signaling pathway activator at a concentration of 0.1 μM. Insome embodiments, the RA signaling pathway activator comprises RA. Insome embodiments, the Pdx1-positive, NKX6-1-positive pancreaticprogenitor cells are contacted with the γ-secretase inhibitor at aconcentration of between 0.1 μM-10 μM. In some embodiments, thePdx1-positive, NKX6-1-positive pancreatic progenitor cells are contactedwith the γ-secretase inhibitor at a concentration of 1 μM. In someembodiments, the γ-secretase inhibitor comprises XXI. In someembodiments, the γ-secretase inhibitor comprises DAPT. In someembodiments, the Pdx1-positive, NKX6-1-positive pancreatic progenitorcells are contacted with the at least one growth factor from the EGFfamily at a concentration of between 2 ng/mL-200 ng/mL. In someembodiments, the Pdx1-positive, NKX6-1-positive pancreatic progenitorcells are contacted with the at least one growth factor from the EGFfamily at a concentration of 20 ng/mL. In some embodiments, the at leastone growth factor from the EGF family comprises betacellulin. In someembodiments, the at least one growth factor from the EGF familycomprises EGF. In some embodiments, the Pdx1-positive, NKX6-1-positivepancreatic progenitor cells are not contacted with the protein kinaseinhibitor. In some embodiments, the Pdx1-positive, NKX6-1-positivepancreatic progenitor cells are contacted with the protein kinaseinhibitor. In some embodiments, the Pdx1-positive, NKX6-1-positivepancreatic progenitor cells are contacted with the protein kinaseinhibitor at a concentration of between 10 nM-1 μM. In some embodiments,the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells arecontacted with the protein kinase inhibitor at a concentration of 100nM. In some embodiments, the protein kinase inhibitor comprisesstaurosporine. In some embodiments, the method includes exposing thepopulation of cells to glucose. In some embodiments, the population ofcells is exposed to glucose at a concentration of between 1 mM-50 mM. Insome embodiments, the population of cells is exposed to glucose at aconcentration of 25 mM. In some embodiments, the conditions that promotecell clustering comprise suspension culture. In some embodiments, thePdx1-positive, NKX6-1-positive pancreatic progenitor cells aremaintained in suspension culture for a period of time sufficient toinduce the differentiation of at least some of the Pdx1-positive,NKX6-1-positive pancreatic progenitor cells into Pdx1-positive,NKX6-1-positive, insulin-positive endocrine cells. In some embodiments,the period of time is at least 7 days. In some embodiments, thesuspension culture is replenished every other day. In some embodiments,at least 15% of the Pdx1-positive, NKX6-1-positive pancreatic progenitorcells are induced to differentiate into Pdx1-positive, NKX6-1-positive,insulin-positive endocrine cells. In some embodiments, at least 99% ofthe Pdx1-positive, NKX6-1-positive pancreatic progenitor cells areinduced to differentiate into Pdx1-positive, NKX6-1-positive,insulin-positive endocrine cells.

In some embodiments, the Pdx1-positive, NKX6-1-positive pancreaticprogenitor cells are obtained by contacting Pdx1-positive pancreaticprogenitor cells under conditions that promote cell clustering with i)at least one growth factor from the FGF family, ii) at least one SHHpathway inhibitor, and optionally iii) low concentrations of a RAsignaling pathway activator, for a period of five days to induce thedifferentiation of at least some of the Pdx1-positive pancreaticprogenitor cells into Pdx1-positive, NKX6-1-positive pancreaticprogenitor cells, wherein the Pdx1-positive, NKX6-1-positive pancreaticprogenitor cells expresses Pdx1 and NKX6-1.

In some embodiments, the Pdx1-positive pancreatic progenitor cells arecontacted with the at least one growth factor from the FGF family at aconcentration of between 1 ng/mL-100 ng/mL. In some embodiments, thePdx1-positive pancreatic progenitor cells are contacted with the atleast one growth factor from the FGF family at a concentration of 50ng/mL. In some embodiments, the at least one growth factor from the FGFfamily comprises keratinocyte growth factor (KGF). In some embodiments,the at least one growth factor from the FGF family is selected from thegroup consisting of FGF2, FGF8B, FGF10, and FGF21. In some embodiments,the Pdx1-positive pancreatic progenitor cells are contacted with the atleast one SHH pathway inhibitor at a concentration of between 0.1 μM and0.5 μM. In some embodiments, the Pdx1-positive pancreatic progenitorcells are contacted with the at least one SHH pathway inhibitor at aconcentration of 0.25 μM. In some embodiments, the at least one SHHpathway inhibitor comprises Sant1. In some embodiments, thePdx1-positive pancreatic progenitor cells are contacted with the RAsignaling pathway activator at a concentration of between 0.01 μM-1.0μM. In some embodiments, the Pdx1-positive pancreatic progenitor cellsare contacted with the RA signaling pathway activator at a concentrationof 0.1 μM. In some embodiments, the RA signaling pathway activatorcomprises RA. In some embodiments, the method includes contacting thePdx1-positive pancreatic progenitor cells with at least one growthfactor from the EGF family. In some embodiments, the Pdx1-positivepancreatic progenitor cells are contacted with the at least one growthfactor from the EGF family at a concentration of between 2 ng/mL-200ng/mL. In some embodiments, the Pdx1-positive pancreatic progenitorcells are contacted with the at least one growth factor from the EGFfamily at a concentration of 20 ng/mL. In some embodiments, the at leastone growth factor from the EGF family comprises betacellulin. In someembodiments, the at least one growth factor from the EGF familycomprises EGF. In some embodiments, the Pdx1-positive pancreaticprogenitor cells are cultured in a suitable culture medium. In someembodiments, the conditions that promote cell clustering comprisesuspension culture. In some embodiments, the suspension culture isreplenished every other day. In some embodiments, an activator ofprotein kinase C is not added to the suspension culture during the 5days. In some embodiments, an activator of protein kinase C is removedfrom the suspension culture prior to the 5 days. In some embodiments,the activator of protein kinase C comprises PdbU. In some embodiments, aBMP signaling pathway inhibitor is not added to the suspension cultureduring the 5 days. In some embodiments, a BMP signaling pathwayinhibitor is removed from the suspension culture prior to the 5 days. Insome embodiments, the BMP signaling pathway inhibitor comprisesLDN193189. In some embodiments, at least 10% of the Pdx1-positivepancreatic progenitor cells in the population are induced todifferentiate into Pdx1-positive, NKX6-1-positive pancreatic progenitorcells. In some embodiments, at least 95% of the Pdx1-positive pancreaticprogenitor cells are induced to differentiate into Pdx1-positive,NKX6-1-positive pancreatic progenitor cells.

In some aspects, the disclosure provides a method of generating SC-βcells from pluripotent cells, the method comprising: a) differentiatingpluripotent stem cells in a population into Pdx1-positive pancreaticprogenitor cells; b) differentiating at least some of the Pdx1-positivepancreatic progenitor cells into Pdx1-positive, NKX6-1-positivepancreatic progenitor cells by a process of contacting the Pdx1-positivepancreatic progenitor cells under conditions that promote cellclustering with i) at least one growth factor from the FGF family, ii)at least one SHH pathway inhibitor, and optionally iii) a RA signalingpathway activator, every other day for a period of five days to inducethe differentiation of at least some of the Pdx1-positive pancreaticprogenitor cells in the population into NKX6-1-positive pancreaticprogenitor cells, wherein the NKX6-1-positive pancreatic progenitorcells expresses Pdx1 and NKX6-1; c) differentiating at least some of thePdx1-positive, NKX6-1-positive pancreatic progenitor cells intoPdx1-positive, NKX6-1-positive, insulin-positive endocrine cells by aprocess of contacting the Pdx1-positive, NKX6-1-positive pancreaticprogenitor cells under conditions that promote cell clustering with i) aTGF-β signaling pathway inhibitor, b) a TH signaling pathway activator,and optionally c) at least one SHH pathway inhibitor, ii) a RA signalingpathway activator, iii) a γ-secretase inhibitor, and vi) at least onegrowth factor from the epidermal growth factor (EGF) family, every otherday for a period of between five and seven days to induce thedifferentiation of at least some of the Pdx1-positive, NKX6-1-positivepancreatic progenitor cells into Pdx1-positive, NKX6-1, insulin-positiveendocrine cells, wherein the Pdx1-positive, NKX6-1, insulin-positiveendocrine cells express Pdx1, NKX6-1, NKX2-2, Mafb, glis3, Sur, Kir6.2,Znt8, SLC2A1, SLC2A3 and/or insulin; and d) differentiating at leastsome of the Pdx1-positive, NKX6-1-positive, insulin-positive endocrinecells into SC-β cells by a process of contacting the Pdx1-positive,NKX6-1-positive, insulin-positive endocrine cells under conditions thatpromote cell clustering with i) a transforming growth factor β (TGF-β)signaling pathway inhibitor, ii) a thyroid hormone signaling pathwayactivator, and optionally iii) a protein kinase inhibitor, every otherday for a period of between seven and 14 days to induce the in vitromaturation of at least some of the Pdx1-positive, NKX6-1-positive,insulin-positive endocrine cells into SC-β cells, wherein the SC-β cellsexhibit a GSIS response in vitro and/or in vivo.

In some embodiments, the disclosure provides a method of generating SC-βcells from pluripotent cells, the method comprising: a) differentiatingat least some pluripotent cells in a population into Pdx1-positivepancreatic progenitor cells; b) differentiating at least some of thePdx1-positive pancreatic progenitor cells into Pdx1-positive,NKX6-1-positive pancreatic progenitor cells by a process of contactingthe Pdx1-positive pancreatic progenitor cells under conditions thatpromote cell clustering with i) KGF, ii) Sant1, and optionally iii) lowconcentrations of RA, every other day for a period of five days toinduce the differentiation of at least one Pdx1-positive pancreaticprogenitor cell in the population into NKX6-1-positive pancreaticprogenitor cells, wherein the NKX6-1-positive pancreatic progenitorcells expresses Pdx1 and NKX6-1; c) differentiating at least some of thePdx1-positive, NKX6-1-positive pancreatic progenitor cells intoPdx1-positive, NKX6-1-positive, insulin-positive endocrine cells by aprocess of contacting the Pdx1-positive, NKX6-1-positive pancreaticprogenitor cells with i) Alk5 Inhibitor II, ii) T3, and optionally iii)Sant1, iv) RA, v) XXI, and vi) betacellulin, every other day for aperiod of between five and seven days to induce the differentiation ofat least some of the Pdx1-positive, NKX6-1-positive pancreaticprogenitor cells into Pdx1-positive, NKX6-1, insulin-positive endocrinecells, wherein the Pdx1-positive, NKX6-1, insulin-positive endocrinecells express Pdx1, NKX6-1, NKX2-2, Mafb, glis3, Sur, Kir6.2, Znt8,SLC2A1, SLC2A3 and/or insulin; and d) differentiating at least some ofthe Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cellsinto SC-β cells by a process of contacting the Pdx1-positive,NKX6-1-positive, insulin-positive endocrine cells under conditions thatpromote cell clustering with i) Alk5 inhibitor II, ii) T3, andoptionally iii) staurosporine, every other day for a period of betweenseven and 14 days to induce the in vitro maturation of at least some ofthe Pdx1-positive, NKX6-1-positive, insulin-producing endocrine cellsinto SC-β cells, wherein the SC-β cells exhibit a GSIS response in vitroand/or in vivo.

In some aspects, the disclosure provides an artificial islet comprisingSC-β cells differentiated in vitro from pluripotent stem cells.

In some aspects, the disclosure provides an artificial pancreascomprising SC-β cells differentiated in vitro from pluripotent stemcells.

The practice of the present invention will typically employ, unlessotherwise indicated, conventional techniques of cell biology, cellculture, molecular biology, transgenic biology, microbiology,recombinant nucleic acid (e.g., DNA) technology, immunology, and RNAinterference (RNAi) which are within the skill of the art. Non-limitingdescriptions of certain of these techniques are found in the followingpublications: Ausubel, F., et al., (eds.), Current Protocols inMolecular Biology, Current Protocols in Immunology, Current Protocols inProtein Science, and Current Protocols in Cell Biology, all John Wiley &Sons, N.Y., edition as of December 2008; Sambrook, Russell, andSambrook, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, 2001; Harlow, E. and Lane,D., Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, 1988; Freshney, R. I., “Culture of Animal Cells, AManual of Basic Technique”, 5th ed., John Wiley & Sons, Hoboken, N.J.,2005. Non-limiting information regarding therapeutic agents and humandiseases is found in Goodman and Gilman's The Pharmacological Basis ofTherapeutics, 11th Ed., McGraw Hill, 2005, Katzung, B. (ed.) Basic andClinical Pharmacology, McGraw-Hill/Appleton & Lange; 10th ed. (2006) or11th edition (July 2009). Non-limiting information regarding genes andgenetic disorders is found in McKusick, V. A.: Mendelian Inheritance inMan. A Catalog of Human Genes and Genetic Disorders. Baltimore: JohnsHopkins University Press, 1998 (12th edition) or the more recent onlinedatabase: Online Mendelian Inheritance in Man, OMIM™. McKusick-NathansInstitute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.)and National Center for Biotechnology Information, National Library ofMedicine (Bethesda, Md.), as of May 1, 2010, available on the worldwideweb at subdomain ncbi.nlm.nih.gov/omim/ and in Online MendelianInheritance in Animals (OMIA), a database of genes, inherited disordersand traits in animal species (other than human and mouse), on theworldwide web at subdomain omia.angis.org.au/contact.shtml. All patents,patent applications, and other publications (e.g., scientific articles,books, websites, and databases) mentioned herein are incorporated byreference in their entirety. In case of a conflict between thespecification and any of the incorporated references, the specification(including any amendments thereof, which may be based on an incorporatedreference), shall control. Standard art-accepted meanings of terms areused herein unless indicated otherwise. Standard abbreviations forvarious terms are used herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A and 1B show comparisons between a previously published controldifferentiation method and a new directed differentiation method. FIG.1A shows a schematic comparing an exemplary directed differentiationmethod of the disclosure for generating INS+ cells from hPSC compared toa previously published control differentiation method. FIG. 1Billustrates histological sections of HUES8 undifferentiated (top),differentiated to DE (middle), and differentiated to PP1 (bottom) andstained with OCT4, SOX17, and PDX1, respectively using a previouslypublished control differentiation method. Scale bar=100 μM.

FIGS. 2A, 2B and 2C demonstrate that stem cell-derived β (SC-β) cellsgenerated in vitro secrete insulin in response to multiple sequentialhigh glucose challenges like primary human β cells. FIGS. 2A, 2B, and 2Care graphs showing ELISA measurements of secreted human insulin fromSC-β (FIG. 2A), primary β cells (FIG. 2B), and PH cells (FIG. 2C)challenged sequentially with 2, 20, 2, 20, 2, and 20 mM glucose. Aftersequential low/high glucose challenges, cells were depolarized with 30mM KCl.

FIGS. 3A, 3B, and 3C demonstrate additional biological replicates of invitro-derived SC-β cells that secrete insulin in response to multiplesequential high glucose challenges like primary β cells. The left panelsare the same as in FIG. 2. Cells SC-β cells (SC-β; FIG. 3A), primary βcells (1°β; FIG. 3B), and PH cells (FIG. 3C) were challengedsequentially with 2, 20, 2, 20, 2, and 20 mM glucose and 30 mM KCl andhuman insulin measured with ELISA.

FIGS. 4A, 4B, 4C, 4D and 4E demonstrate that SC-β cells flux cytosolicCa²⁺ in response to multiple sequential high glucose challenges likeprimary β cells. FIG. 4A is a schematic representation of populationlevel and single cell level detection of cytosolic Ca²⁺ using Fluo-4 AMstaining. Population level measurements were taken on individual wholeclusters (marked by large red circle in schematic), and individual cellswithin intact clusters (marked by small red circles) were analyzed forsingle cell analysis. FIG. 4B is a graph showing population measurementsof dynamic normalized Fluo-4 fluorescence intensity for SC-β cells,primary β cells, and PH cells challenged sequentially with 2, 20, 2, 20,2, and 20 mM glucose and 30 mM KCl. FIG. 4C shows fluorescence images ofFluo-4 AM staining used in single cell analysis. FIG. 4D showsrepresentative images indicating location of single cells that respondedto 3 (yellow), 2 (orange), 1 (blue), and 0 (red) glucose challenges.FIG. 4E shows graphical quantification of the frequency of SC-β cells(n=156), primary β cells (n=114), and PH cells (n=138) that responded to20 mM glucose. Scale bar=100 μm.

FIGS. 5A, 5B, 5C, 5D, 5E and 5F demonstrate that SC-β express human βcell markers at protein and gene expression level. FIG. 5A showsimmunohistochemistry images of cells stained for C-peptide (green),NKX6-1 (red), and somatostatin (grey). FIG. 5B showsimmunohistochemistry images of cells stained for C-peptide (green) andPDX1 (red). FIG. 5C shows immunohistochemistry images of cells stainedfor C-peptide (green) and glucagon (red) with the corresponding DAPIstain (blue). FIG. 5D shows representative flow cytometry dot plots andpopulation percentages of cells stained for C-peptide and NKX6-1. FIG.5E shows hierarchal clustering analysis based on all genes measured bymicroarray of undifferentiated HUES8, PH cells, fetal β cells, and adultprimary β cells sorted for INS (data from Hrvatin et al. (Hrvatin etal., 2014)), and SC-β cells (SC-β) sorted for INS and NKX6-1. FIG. 5Fshows a heat map of the 100 genes with the most variance across allsamples. CP=C-peptide, SST=somatostatin, GCG=glucagon. Scale bar-100 μm.

FIG. 6 illustrates the histology of a SC-β cell cluster stained for DAPI(blue), insulin (green), C-peptide (red). Scale bar=100 μm.

FIGS. 7A, 7B, and 7C demonstrate additional histological staining ofSC-β cells. FIG. 7A illustrates staining for C-peptide (green) and ISL1(red). FIG. 7B illustrates staining for C-peptide (green) and MAFA(red). FIG. 7C illustrates staining for C-peptide (green) and MAFB(red). Scale bar=100 μm.

FIGS. 8A, 8B and 8C show representative flow cytometry dot plots andpopulation percentages of SC-β cells and PH cells stained for C-peptideand SST (FIG. 8A), C-peptide and GCG (FIG. 8B), and SST and GCG (FIG.8C).

FIGS. 9A, 9B and 9C demonstrate that SC-β cell granules are structurallysimilar to primary human β cell granules. FIG. 9A shows electronmicroscopy images of granules highlighting representative crystallizedinsulin granules (red), early insulin granules (yellow), and mixedendocrine granules (blue). Scale bar=500 nm. FIG. 9B shows highermagnification images of granules highlighted in (FIG. 9A). Scale bar=500nm. FIG. 9C shows electron microscopy images of cells labeled withimmunogold staining showing granules that contain insulin (smaller 5 nmblack dots) and/or glucagon (larger 15 nm black dots). Representativeimmunogold particles are highlighted with red arrows (insulin) and bluearrows (glucagon). Scale bar=100 nm.

FIGS. 10A and 10B demonstrate that stem cell-derived β (SC-β) cellsgenerated from hiPSC in vitro secrete insulin in response to multiplesequential high glucose challenges like primary human β cells. FIGS. 10Aand 10B are graphs showing ELISA measurements of secreted human insulinfrom SC-β generated from non-diabetic cells (FIG. 10A) and type 1diabetic cells (FIG. 10B) challenged sequentially with 2, 20, 2, 20, 2,and 20 mM glucose.

FIGS. 11A, 11B, 11C, 11D, 11E, and 11F show representative flowcytometry dot plots and population percentages of cells stained forC-peptide and NKX6-1 from multiple hiPSC lines. FIGS. 11A, 11B, and 11Cshow representative flow cytometry dot plots and population percentagesof cells stained for C-peptide and NKX6-1 from non-diabetic hiPSC lines.FIGS. 11D, 11E, and 11F show representative flow cytometry dot plots andpopulation percentages of cells stained for C-peptide and NKX6-1 fromtype 1 diabetic hiPSC lines.

FIGS. 12A, 12B, 12C and 12D demonstrate that transplanted SC-β cellsfunction rapidly in vivo. FIG. 12A is a graph showing ELISA measurementsof human insulin from the serum of individual mice transplanted withSC-β cells (cultured for 1 week in the final in vitro step), primaryhuman β cells (1° β), or PH cells. Measurements were taken before (whitebars) and 30 min after (black bars) a glucose injection of mice twoweeks post-transplantation. FIG. 12B shows immunohistochemistry imagesof cells transplanted in (FIG. 12A) stained with C-peptide (green) andPDX1 (red) to confirm presence of graft. FIG. 12C is a graph showingELISA measurements of human insulin from the serum of individual micetransplanted with pancreatic progenitors. Measurements were taken before(white bars) and 30 min after (black bars) a glucose injection of micetwo weeks post-transplantation. FIG. 12D is a graph showing ELISAmeasurements of human insulin from the serum of individual micetransplanted with SC-β cells cultured for 2 weeks during the final invitro step. Measurements were taken 30 min after (black bars) a glucoseinjection of mice two weeks post-transplantation. nd=not determined.scale bar-100 μm.

FIGS. 13A and 13B illustrate additional histological sections of SC-βcells and PH cells transplanted into mice 2 wk prior. FIG. 13A shows lowmagnification images of grafts stained for DAPI (blue), C-peptide(green), and GCG (red). Scale bar=200 uM. FIG. 13B shows highermagnification images of grafts stained for C-peptide (green) and GCG(red). Scale bar=100 uM.

FIGS. 14A, 14B and 14C demonstrate the use of media at the last step ofdifferentiation to allow SC-β cells to secrete more insulin in vivo.FIG. 14A shows a schematic showing the use of various media in thevarious steps of the differentiation process. FIG. 14B shows that addingadditional factors, such as Sant1, XXI, and SSP, to the CMRL media atthe last step of differentiation generates a better glucose stimulatedinsulin secretion (GSIS) response by SC-β cells as measured bystimulation index between high and low glucose challenges. FIG. 14Cshows that adding additional factors, such as Sant1, XXI, and SSP, tothe CMRL media at the last step of differentiation generates a betterglucose stimulated insulin secretion (GSIS) response by SC-β cells asmeasured by the amount of insulin released.

FIGS. 15A, 15B, 15C, 15D, 15E, 15F, 15G, 15H and 15 demonstratemodifications to the protocol that can enhance survival and quality ofSC-β cells generated. FIG. 15A is a schematic illustration of theprotocol. FIG. 15B shows how more pure NKX6.1+ endocrine clusters can begenerated (FIG. 15B) using the modified protocol. FIG. 15C demonstrateshow the use of a Rock inhibitor at Steps 3-5 can improve cell survival.FIG. 15D demonstrates how the use of Activin A together withNicotinamide can downregulate SOX2 and improve cell survival. FIG. 15Eshows that SOX2 and NKX6-1 are mutually exclusive. FIG. 15F demonstrateshow the use of staurospaurine at Step 6 generates a near pure endocrinepopulation and FIG. 15G demonstrates how the use of staurospaurine atStep 6 generates a higher percentage of NKX6-1/C-peptide+ cells. FIG.15I demonstrates how the use of XXI in combination with Alk5i and T3 atSteps 5-6 increases the NeuroD+ population when compared to the use ofonly Alk5i and T3(FIG. 15H).

FIGS. 16A, 16B, 16C, 16D, 16E, 16F, 16G, 16H and 16I demonstrateclinical utility of SC-β cells as a diabetes therapy or drug discoveryplatform. FIG. 16A is a schematic illustration of the utility of SC-βcells for treating diabetes or screening drugs to improve function orreplication. FIG. 16B is a table listing diabetic drugs investigated andtheir general therapeutic category. FIG. 16C is a graph showing ELISAmeasurements of secreted human insulin from plated SC-β cells treatedwith the indicated drugs in 2 and 20 mM glucose. Indicated p valuescompare the insulin value in 20 mM glucose between the drug and thecontrol. FIG. 16D is an immunofluorescence image of dispersed and platedSC-β cells stained with DAPI (blue), C-peptide (green), and Ki67 (red)without treatment. FIG. 16E is an immunofluorescence image of dispersedand plated SC-β cells stained with DAPI (blue), C-peptide (green), andKi67 (red) treated with prolactin for 48 hours. FIG. 16F shows agraphical quantification of the fraction of cells that co-expressC-peptide and Ki67. *p<0.05. FIG. 16G is a graph illustrating fastingblood glucose measurements of Akita mice transplanted with SC-β cells(n=6) or PH cells (n=6). *p<0.05 comparing the two cell groups on thesame day. FIG. 16H is a graph illustrating blood glucose measurementsfrom progressively diabetic Akita mice transplanted with SC-β cells orPH cells. Measurements were taken before (white bars) and 20 min after(black bars) a glucose injection of mice transplanted 2 weeks prior.Glucose measurements were saturated at 550 mg/dL. *p<0.05 comparing thetwo cell groups at the same time post glucose injection. FIG. 16I is agraph showing ELISA measurements of human insulin from the serum ofAkita mice 20 min after a glucose injection. Mice were challenged withglucose 2 weeks post transplantation. *p<0.05 comparing the two cellgroups. Scale bar-50 μm.

FIG. 17 is a graph illustrating the body weight of Akita micetransplanted with SC-β cells (n=6) or PH cells (n=6). *p<0.05 comparingthe two cell groups at the 18 and 28 d time point.

DETAILED DESCRIPTION OF THE INVENTION

Aspects of the disclosure relate to compositions, methods, kits, andagents for generating stem cell-derived β (SC-β) cells (e.g., maturepancreatic β cells) from at least one insulin-positive endocrine cell ora precursor thereof (e.g., iPS cells, hESCs, definitive endoderm cells,primitive gut tube cells, Pdx1-positive pancreatic progenitor cells,Pdx1-positive, NKX6-1-positive pancreatic progenitor cells,Ngn3-positive endocrine progenitor cells, etc.), and SC-β cells producedby those compositions, methods, kits, and agents for use in celltherapies, assays (e.g., drug screening), and various methods oftreatment.

In addition, aspects of the disclosure relate to methods ofidentification of the SC-β cells that are detectable based onmorphological criteria, without the need to employ a selectable marker,as well as functional characteristics, such as ability to expressinsulin, secrete insulin in response to one or more glucose challenges,exhibit a mature GSIS response, and organize in islets in pancreas invivo, and typically have small spindle like cells of about 9-15 mdiameter.

In addition, aspects of the disclosure relate to methods of identifyingβ cell maturation factors. One of skill in the art will be aware of, orwill readily be able to ascertain, whether a particular β cellmaturation factor is functional using assays known in the art. Forexample, the ability of a β cell maturation factor to convert at leastone insulin-positive endocrine cell or a precursor thereof to a SC-βcell can be assessed using the assays as disclosed herein in. Otherconvenient assays include measuring the ability to activatetranscription of a reporter construct containing a β cell marker bindingsite operably linked to a nucleic acid sequence encoding a detectablemarker such as luciferase. One assay involves determining whether thecandidate β cell maturation factor induces at least one insulin-positiveendocrine cell to become a SC-β cell or express markers of a β cell orexhibit functional characteristics of a mature β cell as disclosedherein. Determination of such expression of β cell markers can bedetermined using any suitable method, e.g., immunoblotting. Such assaysmay readily be adapted to identify or confirm activity of agents thatdirectly convert at least one insulin-positive endocrine cell or aprecursor thereof to a SC-β cell.

The in vitro-matured, SC-β cells (i.e., pancreatic β cells) generatedaccording to the inventive methods described herein demonstrate manyadvantages, for example, they perform glucose stimulated insulinsecretion in vitro, resemble human islet β cells by gene expression andultrastructure, secrete human insulin and ameliorate hyperglycemia whentransplanted into mice, provide a new platform for cell therapy (e.g.,transplantation into a subject in need of additional and/or functional βcells), drug screening (e.g., for insulin production/secretion,survival, dedifferentiation, etc.), research (e.g., determining thedifferences in function between normal and diabetic β cells), and tissueengineering (e.g., using the SC-β cells as the first cell type inreconstructing an islet).

Definitions

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected here. Unless otherwisedefined, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs.

The term “differentiated cell” is meant any primary cell that is not, inits native form, pluripotent as that term is defined herein. Statedanother way, the term “differentiated cell” refers to a cell of a morespecialized cell type derived from a cell of a less specialized celltype (e.g., a stem cell such as an induced pluripotent stem cell) in acellular differentiation process. Without wishing to be limited totheory, a pluripotent stem cell in the course of normal ontogeny candifferentiate first to an endoderm cell that is capable of formingpancreas cells and other endoderm cell types. Further differentiation ofan endoderm cell leads to the pancreatic pathway, where ^(˜)98% of thecells become exocrine, ductular, or matrix cells, and ^(˜)2% becomeendocrine cells. Early endocrine cells are islet progenitors, which canthen differentiate further into insulin-producing cells (e.g. functionalendocrine cells) which secrete insulin, glucagon, somatostatin, orpancreatic polypeptide. Endoderm cells can also be differentiate intoother cells of endodermal origin, e.g. lung, liver, intestine, thymusetc.

As used herein, the term “somatic cell” refers to any cells forming thebody of an organism, as opposed to germline cells. In mammals, germlinecells (also known as “gametes”) are the spermatozoa and ova which fuseduring fertilization to produce a cell called a zygote, from which theentire mammalian embryo develops. Every other cell type in the mammalianbody—apart from the sperm and ova, the cells from which they are made(gametocytes) and undifferentiated stem cells—is a somatic cell:internal organs, skin, bones, blood, and connective tissue are all madeup of somatic cells. In some embodiments the somatic cell is a“non-embryonic somatic cell”, by which is meant a somatic cell that isnot present in or obtained from an embryo and does not result fromproliferation of such a cell in vitro. In some embodiments the somaticcell is an “adult somatic cell”, by which is meant a cell that ispresent in or obtained from an organism other than an embryo or a fetusor results from proliferation of such a cell in vitro. Unless otherwiseindicated the methods for converting at least one insulin-positiveendocrine cell or precursor thereof to an insulin-producing, glucoseresponsive cell can be performed both in vivo and in vitro (where invivo is practiced when at least one insulin-positive endocrine cell orprecursor thereof are present within a subject, and where in vitro ispracticed using an isolated at least one insulin-positive endocrine cellor precursor thereof maintained in culture).

As used herein, the term “adult cell” refers to a cell found throughoutthe body after embryonic development.

The term “endoderm cell” as used herein refers to a cell which is fromone of the three primary germ cell layers in the very early embryo (theother two germ cell layers are the mesoderm and ectoderm). The endodermis the innermost of the three layers. An endoderm cell differentiates togive rise first to the embryonic gut and then to the linings of therespiratory and digestive tracts (e.g. the intestine), the liver and thepancreas.

The term “a cell of endoderm origin” as used herein refers to any cellwhich has developed or differentiated from an endoderm cell. Forexample, a cell of endoderm origin includes cells of the liver, lung,pancreas, thymus, intestine, stomach and thyroid. Without wishing to bebound by theory, liver and pancreas progenitors (also referred to aspancreatic progenitors) are develop from endoderm cells in the embryonicforegut. Shortly after their specification, liver and pancreasprogenitors rapidly acquire markedly different cellular functions andregenerative capacities. These changes are elicited by inductive signalsand genetic regulatory factors that are highly conserved amongvertebrates. Interest in the development and regeneration of the organshas been fueled by the intense need for hepatocytes and pancreatic βcells in the therapeutic treatment of liver failure and type I diabetes.Studies in diverse model organisms and humans have revealedevolutionarily conserved inductive signals and transcription factornetworks that elicit the differentiation of liver and pancreatic cellsand provide guidance for how to promote hepatocyte and β celldifferentiation from diverse stem and progenitor cell types.

The term “definitive endoderm” as used herein refers to a celldifferentiated from an endoderm cell and which can be differentiatedinto a SC-β cell (e.g., a pancreatic β cell). A definitive endoderm cellexpresses the marker Sox17. Other markers characteristic of definitiveendoderm cells include, but are not limited to MIXL2, GATA4, HNF3b, GSC,FGF17, VWF, CALCR, FOXQ1, CXCR4, Cerberus, OTX2, goosecoid, C-Kit, CD99,CMKOR1 and CRIP1. In particular, definitive endoderm cells hereinexpress Sox17 and in some embodiments Sox17 and HNF3B, and do notexpress significant levels of GATA4, SPARC, APF or DAB. Definitiveendoderm cells are not positive for the marker Pdx1 (e.g. they arePdx1-negative). Definitive endoderm cells have the capacity todifferentiate into cells including those of the liver, lung, pancreas,thymus, intestine, stomach and thyroid. The expression of Sox17 andother markers of definitive endoderm may be assessed by any method knownby the skilled person such as immunochemistry, e.g., using an anti-Sox17antibody, or quantitative RT-PCR.

The term “pancreatic endoderm” refers to a cell of endoderm origin whichis capable of differentiating into multiple pancreatic lineages,including pancreatic β cells, but no longer has the capacity todifferentiate into non-pancreatic lineages.

The term “primitive gut tube cell” or “gut tube cell” as used hereinrefers to a cell differentiated from an endoderm cell and which can bedifferentiated into a SC-β cell (e.g., a pancreatic β cell). A primitivegut tube cell expresses at least one of the following markers: HNF1-β,HNF3-β or HNF4-α. Primitive gut tube cells have the capacity todifferentiate into cells including those of the lung, liver, pancreas,stomach, and intestine. The expression of HNF1-β and other markers ofprimitive gut tube may be assessed by any method known by the skilledperson such as immunochemistry, e.g., using an anti-HNF1-β antibody.

The term “pancreatic progenitor”, “pancreatic endocrine progenitor”,“pancreatic precursor” or “pancreatic endocrine precursor” are usedinterchangeably herein and refer to a stem cell which is capable ofbecoming a pancreatic hormone expressing cell capable of formingpancreatic endocrine cells, pancreatic exocrine cells or pancreatic ductcells. These cells are committed to differentiating towards at least onetype of pancreatic cell, e.g. beta cells that produce insulin; alphacells that produce glucagon; delta cells (or D cells) that producesomatostatin; and/or F cells that produce pancreatic polypeptide. Suchcells can express at least one of the following markers: NGN3, NKX2.2,NeuroD, ISL-1, Pax4, Pax6, or ARX.

The term “pdx1-positive pancreatic progenitor” as used herein refers toa cell which is a pancreatic endoderm (PE) cell which has the capacityto differentiate into SC-β cells, such as pancreatic β cells. APdx1-positive pancreatic progenitor expresses the marker Pdx1. Othermarkers include, but are not limited to Cdcp1, or Ptf1a, or HNF6 orNRx2.2. The expression of Pdx1 may be assessed by any method known bythe skilled person such as immunochemistry using an anti-Pdx1 antibodyor quantitative RT-PCR.

The term “pdx1-positive, NKX6-1-positive pancreatic progenitor” as usedherein refers to a cell which is a pancreatic endoderm (PE) cell whichhas the capacity to differentiate into insulin-producing cells, such aspancreatic β cells. A pdx1-positive, NKX6-1-positive pancreaticprogenitor expresses the markers Pdx1 and NKX6-1. Other markers include,but are not limited to Cdcp1, or Ptf1a, or HNF6 or NRx2.2. Theexpression of NKX6-1 may be assessed by any method known by the skilledperson such as immunochemistry using an anti-NKX6-1 antibody orquantitative RT-PCR.

The term “Ngn3-positive endocrine progenitor” as used herein refers toprecursors of pancreatic endocrine cells expressing the transcriptionfactor Neurogenin-3 (Ngn3). Progenitor cells are more differentiatedthan multipotent stem cells and can differentiate into only few celltypes. In particular, Ngn3-positive endocrine progenitor cells have theability to differentiate into the five pancreatic endocrine cell types(α, β, δ, ε and PP). The expression of Ngn3 may be assessed by anymethod known by the skilled person such as immunochemistry using ananti-Ngn3 antibody or quantitative RT-PCR.

The terms “NeuroD” and “NeuroD1” are used interchangeably and identify aprotein expressed in pancreatic endocrine progenitor cells and the geneencoding it.

The terms “insulin-positive β-like cell” and “insulin-positive endocrinecell” refer to cells (e.g., pancreatic endocrine cells) that displays atleast one marker indicative of a pancreatic β cell and also expressesinsulin but lack a GSIS response characteristic of an endogenous β cell.

A “precursor thereof” as the term relates to an insulin-positiveendocrine cell refers to any cell that is capable of differentiatinginto an insulin-positive endocrine cell, including for example, apluripotent stem cell, a definitive endoderm cell, a primitive gut tubecell, a pancreatic progenitor cell, or endocrine progenitor cell, whencultured under conditions suitable for differentiating the precursorcell into the insulin-positive endocrine cell.

The terms “stem cell-derived β cell”, “SC-β cell”, “functional β cell”,“functional pancreatic β cell” and “mature SC-β cell” refer to cells(e.g., pancreatic β cells) that display at least one marker indicativeof a pancreatic β cell (e.g., PDX-1 or NKX6-1), expresses insulin, anddisplay a GSIS response characteristic of an endogenous mature β cell.In some embodiments, the “SC-β cell” comprises a mature pancreatic βcells. It is to be understood that the SC-β cells need not be derived(e.g., directly) from stem cells, as the methods of the disclosure arecapable of deriving SC-β cells from any insulin-positive endocrine cellor precursor thereof using any cell as a starting point (e.g., one canuse embryonic stem cells, induced-pluripotent stem cells, progenitorcells, partially reprogrammed somatic cells (e.g., a somatic cell whichhas been partially reprogrammed to an intermediate state between aninduced pluripotent stem cell and the somatic cell from which it wasderived), multipotent cells, totipotent cells, a transdifferentiatedversion of any of the foregoing cells, etc, as the invention is notintended to be limited in this manner). In some embodiments, the SC-βcells exhibit a response to multiple glucose challenges (e.g., at leastone, at least two, or at least three or more sequential glucosechallenges). In some embodiments, the response resembles the response ofendogenous islets (e.g., human islets) to multiple glucose challenges.In some embodiments, the morphology of the SC-β cell resembles themorphology of an endogenous β cell. In some embodiments, the SC-β cellexhibits an in vitro GSIS response that resembles the GSIS response ofan endogenous β cell. In some embodiments, the SC-β cell exhibits an invivo GSIS response that resembles the GSIS response of an endogenous βcell. In some embodiments, the SC-β cell exhibits both an in vitro andin vivo GSIS response that resembles the GSIS response of an endogenousβ cell. The GSIS response of the SC-β cell can be observed within twoweeks of transplantation of the SC-β cell into a host (e.g., a human oranimal). In some embodiments, the SC-β cells package insulin intosecretory granules. In some embodiments, the SC-β cells exhibitencapsulated crystalline insulin granules. In some embodiments, the SC-βcells exhibit a stimulation index of greater than 1. In someembodiments, the SC-β cells exhibit a stimulation index of greater than1.1. In some embodiments, the SC-β cells exhibit a stimulation index ofgreater than 2. In some embodiments, the SC-β cells exhibitcytokine-induced apoptosis in response to cytokines. In someembodiments, insulin secretion from the SC-β cells is enhanced inresponse to known antidiabetic drugs (e.g., secretagogues). In someembodiments, the SC-β cells are monohormonal. In some embodiments, theSC-β cells do not abnormally co-express other hormones, such asglucagon, somatostatin or pancreatic polypeptide. In some embodiments,the SC-β cells exhibit a low rate of replication. In some embodiments,the SC-β cells increase intracellular Ca²⁺ in response to glucose. Theterm “exocrine cell” as used herein refers to a cell of an exocrinegland, i.e. a gland that discharges its secretion via a duct. Inparticular embodiments, an exocrine cells refers to a pancreaticexocrine cell, which is a pancreatic cell that produces enzymes that aresecreted into the small intestine. These enzymes help digest food as itpasses through the gastrointestinal tract. Pancreatic exocrine cells arealso known as islets of Langerhans, that secrete two hormones, insulinand glucagon. A pancreatic exocrine cell can be one of several celltypes: alpha-2 cells (which produce the hormone glucagon); or β cells(which manufacture the hormone insulin); and alpha-1 cells (whichproduce the regulatory agent somatostatin). Non-insulin-producingexocrine cells as used herein refers to alpha-2 cells or alpha-1 cells.Note, the term pancreatic exocrine cells encompasses “pancreaticendocrine cells” which refer to a pancreatic cell that produces hormones(e.g., insulin (produced from β cells), glucagon (produced by alpha-2cells), somatostatin (produced by delta cells) and pancreaticpolypeptide (produced by F cells) that are secreted into thebloodstream.

As used herein, the term “insulin-producing cell” refers to a celldifferentiated from a pancreatic progenitor, or precursor thereof, whichsecretes insulin. An insulin-producing cell includes pancreatic β cellsas that term is described herein, as well as pancreatic β-like cells(i.e., insulin-positive, endocrine cells) that synthesize (i.e.,transcribe the insulin gene, translate the proinsulin mRNA, and modifythe proinsulin mRNA into the insulin protein), express (i.e., manifestthe phenotypic trait carried by the insulin gene), or secrete (releaseinsulin into the extracellular space) insulin in a constitutive orinducible manner. A population of insulin-producing cells e.g. producedby differentiating insulin-positive, endocrine cells or a precursorthereof into SC-β cells according to the methods of the presentinvention can be pancreatic β cells or β-like cells (e.g., cells thathave at least one, or at least two least two) characteristic of anendogenous β cell and exhibit a GSIS response that resembles anendogenous adult β cell. The novelty of the present composition andmethods is not negated by the presence of cells in the population thatproduce insulin naturally (e.g., β cells). It is also contemplated thatthe population of insulin-producing cells, e.g. produced by the methodsas disclosed herein can comprise mature pancreatic β cells or SC-βcells, and can also contain non-insulin-producing cells (i.e. cells of βcell like phenotype with the exception they do not produce or secreteinsulin).

As used herein, the terms “endogenous β cell”, “endogenous maturepancreatic β cell” or “endogenous pancreatic β cell” refer to aninsulin-producing cell of the pancreas or a cell of a pancreatic β cell(β cell) phenotype. The phenotype of a pancreatic β cell is well knownby persons of ordinary skill in the art, and include, for example,secretion of insulin in response to an increase in glucose level,expression of markers such as c-peptide, Pdx1 polypeptide and Glut 2, aswell as distinct morphological characteristics such as organized inislets in pancreas in vivo, and typically have small spindle like cellsof about 9-15 m diameter.

The term “SC-β cell”, “pancreatic β-like cell”, and “mature pancreaticβ-like” as used herein refer to cells produced by the methods asdisclosed herein which expresses at least 15% of the amount of insulinexpressed by an endogenous pancreatic β cell, or at least about 20% orat least about 30%, or at least about 40%, or at least about 50%, or atleast about 60%, or at least about 70%, or at least about 80%, or atleast about 90%, or at least about 100% or greater than 100%, such as atleast about 1.5-fold, or at least about 2-fold, or at least about2.5-fold, or at least about 3-fold, or at least about 4-fold or at leastabout 5-fold or more than about 5-fold the amount of the insulinsecreted by an endogenous pancreatic β cell, or alternatively exhibitsat least one, or at least two characteristics of an endogenouspancreatic β cell, for example, but not limited to, secretion of insulinin response to glucose, and expression of β cell markers, such as forexample, c-peptide, Pdx1 and glut-2. In one embodiment, the SC-β cell isnot an immortalized cell (e.g. proliferate indefinitely in culture). Inone embodiment, the SC-β cell is not a transformed cell, e.g., a cellthat exhibits a transformation property, such as growth in soft agar, orabsence of contact inhibition.

The term “β cell marker” refers to, without limitation, proteins,peptides, nucleic acids, polymorphism of proteins and nucleic acids,splice variants, fragments of proteins or nucleic acids, elements, andother analytes which are specifically expressed or present in pancreaticβ cells. Exemplary β cell markers include, but are not limited to,pancreatic and duodenal homeobox 1 (Pdx1) polypeptide, insulin,c-peptide, amylin, E-cadherin, Hnf3p, PCI/3, B2, Nkx2.2, NKX6-1, GLUT2,PC2, ZnT-8, Isll, Pax6, Pax4, NeuroD, Hnflb, Hnf-6, Hnf-3beta, and MafA,and those described in Zhang et al., Diabetes. 50(10):2231-6 (2001). Insome embodiment, the β cell marker is a nuclear 3-cell marker. In someembodiments, the β cell marker is Pdx1 or PH3.

The term “pancreatic endocrine marker” refers to without limitation,proteins, peptides, nucleic acids, polymorphism of proteins and nucleicacids, splice variants, fragments of proteins or nucleic acids,elements, and other analytes which are specifically expressed or presentin pancreatic endocrine cells. Exemplary pancreatic endocrine cellmarkers include, but are not limited to, Ngn-3, NeuroD and Islet-1.

The term “non-insulin-producing cell” as used herein is meant any cellof endoderm origin that does not naturally synthesize, express, orsecrete insulin constitutively or by induction. Thus, the term“non-insulin-producing cells” as used herein excludes pancreatic βcells. Examples of non-insulin-producing cells that can be used in themethods of the present invention include pancreatic non-β cells, such asamylase producing cells, acinar cells, cells of ductal adenocarcinomacell lines (e.g., CD18, CD11, and Capan-I cells (see Busik et al., 1997;Schaffert et al. 1997). Non-pancreatic cells of endoderm origin couldalso be used, for example, non-pancreatic stem cells and cells of otherendocrine or exocrine organs, including, for example, liver cells, tymuscells, thyroid cells, intestine cells, lung cells and pituitary cells.In some embodiments, the non-insulin-producing endodermal cells can bemammalian cells or, even more specifically, human cells. Examples of thepresent method using mammalian pancreatic non-islet, pancreatic amylaseproducing cells, pancreatic acinar cells are provided herein.

The term “phenotype” refers to one or a number of total biologicalcharacteristics that define the cell or organism under a particular setof environmental conditions and factors, regardless of the actualgenotype.

The term “pluripotent” as used herein refers to a cell with thecapacity, under different conditions, to differentiate to more than onedifferentiated cell type, and preferably to differentiate to cell typescharacteristic of all three germ cell layers. Pluripotent cells arecharacterized primarily by their ability to differentiate to more thanone cell type, preferably to all three germ layers, using, for example,a nude mouse teratoma formation assay. Pluripotency is also evidenced bythe expression of embryonic stem (ES) cell markers, although thepreferred test for pluripotency is the demonstration of the capacity todifferentiate into cells of each of the three germ layers. It should benoted that simply culturing such cells does not, on its own, render thempluripotent. Reprogrammed pluripotent cells (e.g. iPS cells as that termis defined herein) also have the characteristic of the capacity ofextended passaging without loss of growth potential, relative to primarycell parents, which generally have capacity for only a limited number ofdivisions in culture.

As used herein, the terms “iPS cell” and “induced pluripotent stem cell”are used interchangeably and refers to a pluripotent stem cellartificially derived (e.g., induced or by complete reversal) from anon-pluripotent cell, typically an adult somatic cell, for example, byinducing a forced expression of one or more genes.

The term “progenitor” or “precursor” cell are used interchangeablyherein and refer to cells that have a cellular phenotype that is moreprimitive (i.e., is at an earlier step along a developmental pathway orprogression than is a fully differentiated cell) relative to a cellwhich it can give rise to by differentiation. Often, progenitor cellsalso have significant or very high proliferative potential. Progenitorcells can give rise to multiple distinct differentiated cell types or toa single differentiated cell type, depending on the developmentalpathway and on the environment in which the cells develop anddifferentiate.

The term “stem cell” as used herein, refers to an undifferentiated cellwhich is capable of proliferation and giving rise to more progenitorcells having the ability to generate a large number of mother cells thatcan in turn give rise to differentiated, or differentiable daughtercells. The daughter cells themselves can be induced to proliferate andproduce progeny that subsequently differentiate into one or more maturecell types, while also retaining one or more cells with parentaldevelopmental potential. The term “stem cell” refers to a subset ofprogenitors that have the capacity or potential, under particularcircumstances, to differentiate to a more specialized or differentiatedphenotype, and which retains the capacity, under certain circumstances,to proliferate without substantially differentiating. In one embodiment,the term stem cell refers generally to a naturally occurring mother cellwhose descendants (progeny) specialize, often in different directions,by differentiation, e.g., by acquiring completely individual characters,as occurs in progressive diversification of embryonic cells and tissues.Cellular differentiation is a complex process typically occurringthrough many cell divisions. A differentiated cell may derive from amultipotent cell which itself is derived from a multipotent cell, and soon. While each of these multipotent cells may be considered stem cells,the range of cell types each can give rise to may vary considerably.Some differentiated cells also have the capacity to give rise to cellsof greater developmental potential. Such capacity may be natural or maybe induced artificially upon treatment with various factors. In manybiological instances, stem cells are also “multipotent” because they canproduce progeny of more than one distinct cell type, but this is notrequired for “stem-ness.” Self-renewal is the other classical part ofthe stem cell definition, and it is essential as used in this document.In theory, self-renewal can occur by either of two major mechanisms.Stem cells may divide asymmetrically, with one daughter retaining thestem state and the other daughter expressing some distinct otherspecific function and phenotype. Alternatively, some of the stem cellsin a population can divide symmetrically into two stems, thusmaintaining some stem cells in the population as a whole, while othercells in the population give rise to differentiated progeny only.Formally, it is possible that cells that begin as stem cells mightproceed toward a differentiated phenotype, but then “reverse” andre-express the stem cell phenotype, a term often referred to as“dedifferentiation” or “reprogramming” or “retrodifferentiation” bypersons of ordinary skill in the art. As used herein, the term“pluripotent stem cell” includes embryonic stem cells, inducedpluripotent stem cells, placental stem cells, etc.

In the context of cell ontogeny, the adjective “differentiated”, or“differentiating” is a relative term meaning a “differentiated cell” isa cell that has progressed further down the developmental pathway thanthe cell it is being compared with. Thus, stem cells can differentiateto lineage-restricted precursor cells (such as a mesodermal stem cell),which in turn can differentiate into other types of precursor cellsfurther down the pathway (such as an cardiomyocyte precursor), and thento an end-stage differentiated cell, which plays a characteristic rolein a certain tissue type, and may or may not retain the capacity toproliferate further.

The term “embryonic stem cell” is used to refer to the pluripotent stemcells of the inner cell mass of the embryonic blastocyst (see U.S. Pat.Nos. 5,843,780, 6,200,806). Such cells can similarly be obtained fromthe inner cell mass of blastocysts derived from somatic cell nucleartransfer (see, for example, U.S. Pat. Nos. 5,945,577, 5,994,619,6,235,970). The distinguishing characteristics of an embryonic stem celldefine an embryonic stem cell phenotype. Accordingly, a cell has thephenotype of an embryonic stem cell if it possesses one or more of theunique characteristics of an embryonic stem cell such that that cell canbe distinguished from other cells. Exemplary distinguishing embryonicstem cell characteristics include, without limitation, gene expressionprofile, proliferative capacity, differentiation capacity, karyotype,responsiveness to particular culture conditions, and the like.

The term “adult stem cell” or “ASC” is used to refer to any multipotentstem cell derived from non-embryonic tissue, including fetal, juvenile,and adult tissue. Stem cells have been isolated from a wide variety ofadult tissues including blood, bone marrow, brain, olfactory epithelium,skin, pancreas, skeletal muscle, and cardiac muscle. Each of these stemcells can be characterized based on gene expression, factorresponsiveness, and morphology in culture. Exemplary adult stem cellsinclude neural stem cells, neural crest stem cells, mesenchymal stemcells, hematopoietic stem cells, and pancreatic stem cells. As indicatedabove, stem cells have been found resident in virtually every tissue.Accordingly, the present invention appreciates that stem cellpopulations can be isolated from virtually any animal tissue.

The term “pancreas” refers to a glandular organ that secretes digestiveenzymes and hormones. In humans, the pancreas is a yellowish organ about7 in. (17.8 cm) long and 1.5 in. (3.8 cm) wide. It lies beneath thestomach and is connected to the small intestine, muscular hoselikeportion of the gastrointestinal tract extending from the lower end ofthe stomach (pylorus) to the anal opening. Most of the pancreatic tissueconsists of grapelike clusters of cells that produce a clear fluid(pancreatic juice) that flows into the duodenum through a common ductalong with bile from the liver. Pancreatic juice contains threedigestive enzymes: tryptase, amylase, and lipase, that, along withintestinal enzymes, complete the digestion of proteins, carbohydrates,and fats, respectively. Scattered among the enzyme-producing cells ofthe pancreas are small groups of endocrine cells, called the islets ofLangerhans, that secrete two hormones, insulin and glucagon. Thepancreatic islets contain several types of cells: alpha-2 cells, whichproduce the hormone glucagon; β cells (also referred to herein as“pancreatic β cells”), which manufacture the hormone insulin; andalpha-1 cells, which produce the regulatory agent somatostatin. Thesehormones are secreted directly into the bloodstream, and together, theyregulate the level of glucose in the blood. Insulin lowers the bloodsugar level and increases the amount of glycogen (stored carbohydrate)in the liver; glucagon has the opposite action. Failure of theinsulin-secreting cells to function properly results in diabetes ordiabetes mellitus.

The term “reprogramming” as used herein refers to the process thatalters or reverses the differentiation state of a somatic cell. The cellcan either be partially or terminally differentiated prior to thereprogramming. Reprogramming encompasses complete reversion of thedifferentiation state of a somatic cell to a pluripotent cell. Suchcomplete reversal of differentiation produces an induced pluripotent(iPS) cell. Reprogramming as used herein also encompasses partialreversion of a cells differentiation state, for example to a multipotentstate or to a somatic cell that is neither pluripotent or multipotent,but is a cell that has lost one or more specific characteristics of thedifferentiated cell from which it arises, e.g. direct reprogramming of adifferentiated cell to a different somatic cell type. Reprogramminggenerally involves alteration, e.g., reversal, of at least some of theheritable patterns of nucleic acid modification (e.g., methylation),chromatin condensation, epigenetic changes, genomic imprinting, etc.,that occur during cellular differentiation as a zygote develops into anadult.

The term “agent” as used herein means any compound or substance such as,but not limited to, a small molecule, nucleic acid, polypeptide,peptide, drug, ion, etc. An “agent” can be any chemical, entity ormoiety, including without limitation synthetic and naturally-occurringproteinaceous and non-proteinaceous entities. In some embodiments, anagent is nucleic acid, nucleic acid analogues, proteins, antibodies,peptides, aptamers, oligomer of nucleic acids, amino acids, orcarbohydrates including without limitation proteins, oligonucleotides,ribozymes, DNAzymes, glycoproteins, siRNAs, lipoproteins, aptamers, andmodifications and combinations thereof etc. In certain embodiments,agents are small molecule having a chemical moiety. For example,chemical moieties included unsubstituted or substituted alkyl, aromatic,or heterocyclyl moieties including macrolides, leptomycins and relatednatural products or analogues thereof. Compounds can be known to have adesired activity and/or property, or can be selected from a library ofdiverse compounds.

As used herein, the term “contacting” (i.e., contacting at least oneinsulin-positive endocrine cell or a precursor thereof with a βcellmaturation factor, or combination of β cell maturation factors) isintended to include incubating the β cell maturation factor and the celltogether in vitro (e.g., adding the β cell maturation factors to cellsin culture). In some embodiments, the term “contacting” is not intendedto include the in vivo exposure of cells to the compounds as disclosedherein that may occur naturally in a subject (i.e., exposure that mayoccur as a result of a natural physiological process). The step ofcontacting at least one insulin-positive endocrine cell or a precursorthereof with a β cell maturation factor as in the embodiments related tothe production of SC-β cells can be conducted in any suitable manner.For example, the cells may be treated in adherent culture, or insuspension culture. In some embodiments, the cells are treated inconditions that promote cell clustering. The disclosure contemplates anyconditions which promote cell clustering. Examples of conditions thatpromote cell clustering include, without limitation, suspension culturein low attachment tissue culture plates, spinner flasks, aggrewellplates. In some embodiments, the inventors have observed that clustershave remained stable in media containing 10% serum. In some embodiments,the conditions that promote clustering include a low serum medium.

It is understood that the cells contacted with a cell maturation factorcan also be simultaneously or subsequently contacted with another agent,such as a growth factor or other differentiation agent or environmentsto stabilize the cells, or to differentiate the cells further.

Similarly, at least one insulin-positive endocrine cell or a precursorthereof can be contacted with at least one β cell maturation factor andthen contacted with at least another β cell maturation factor. In someembodiments, the cell is contacted with at least one β cell maturationfactor, and the contact is temporally separated, and in someembodiments, a cell is contacted with at least one β cell maturationfactor substantially simultaneously. In some embodiments, the cell iscontacted with at least two, at least three, at least four, at leastfive, at least six, at least seven, at least eight, at least nine, or atleast 10 β cell maturation factors.

The term “cell culture medium” (also referred to herein as a “culturemedium” or “medium”) as referred to herein is a medium for culturingcells containing nutrients that maintain cell viability and supportproliferation. The cell culture medium may contain any of the followingin an appropriate combination: salt(s), buffer(s), amino acids, glucoseor other sugar(s), antibiotics, serum or serum replacement, and othercomponents such as peptide growth factors, etc. Cell culture mediaordinarily used for particular cell types are known to those skilled inthe art.

The term “cell line” refers to a population of largely or substantiallyidentical cells that has typically been derived from a single ancestorcell or from a defined and/or substantially identical population ofancestor cells. The cell line may have been or may be capable of beingmaintained in culture for an extended period (e.g., months, years, foran unlimited period of time). It may have undergone a spontaneous orinduced process of transformation conferring an unlimited culturelifespan on the cells. Cell lines include all those cell linesrecognized in the art as such. It will be appreciated that cells acquiremutations and possibly epigenetic changes over time such that at leastsome properties of individual cells of a cell line may differ withrespect to each other. In some embodiments, a cell line comprises a SC-βcell described herein.

The term “exogenous” refers to a substance present in a cell or organismother than its native source. For example, the terms “exogenous nucleicacid” or “exogenous protein” refer to a nucleic acid or protein that hasbeen introduced by a process involving the hand of man into a biologicalsystem such as a cell or organism in which it is not normally found orin which it is found in lower amounts. A substance will be consideredexogenous if it is introduced into a cell or an ancestor of the cellthat inherits the substance. In contrast, the term “endogenous” refersto a substance that is native to the biological system.

The term “expression” refers to the cellular processes involved inproducing RNA and proteins and as appropriate, secreting proteins,including where applicable, but not limited to, for example,transcription, translation, folding, modification and processing.“Expression products” include RNA transcribed from a gene andpolypeptides obtained by translation of mRNA transcribed from a gene.

The terms “genetically modified” or “engineered” cell as used hereinrefers to a cell into which an exogenous nucleic acid has beenintroduced by a process involving the hand of man (or a descendant ofsuch a cell that has inherited at least a portion of the nucleic acid).The nucleic acid may for example contain a sequence that is exogenous tothe cell, it may contain native sequences (i.e., sequences naturallyfound in the cells) but in a non-naturally occurring arrangement (e.g.,a coding region linked to a promoter from a different gene), or alteredversions of native sequences, etc. The process of transferring thenucleic into the cell can be achieved by any suitable technique.Suitable techniques include calcium phosphate or lipid-mediatedtransfection, electroporation, and transduction or infection using aviral vector. In some embodiments the polynucleotide or a portionthereof is integrated into the genome of the cell. The nucleic acid mayhave subsequently been removed or excised from the genome, provided thatsuch removal or excision results in a detectable alteration in the cellrelative to an unmodified but otherwise equivalent cell. It should beappreciated that the term genetically modified is intended to includethe introduction of a modified RNA directly into a cell (e.g., asynthetic, modified RNA). Such synthetic modified RNAs includemodifications to prevent rapid degradation by endo- and exo-nucleasesand to avoid or reduce the cell's innate immune or interferon responseto the RNA. Modifications include, but are not limited to, for example,(a) end modifications, e.g., 5′ end modifications (phosphorylationdephosphorylation, conjugation, inverted linkages, etc.), 3′ endmodifications (conjugation, DNA nucleotides, inverted linkages, etc.),(b) base modifications, e.g., replacement with modified bases,stabilizing bases, destabilizing bases, or bases that base pair with anexpanded repertoire of partners, or conjugated bases, (c) sugarmodifications (e.g., at the 2′ position or 4′ position) or replacementof the sugar, as well as (d) internucleoside linkage modifications,including modification or replacement of the phosphodiester linkages. Tothe extent that such modifications interfere with translation (i.e.,results in a reduction of 50% or more in translation relative to thelack of the modification—e.g., in a rabbit reticulocyte in vitrotranslation assay), the modification is not suitable for the methods andcompositions described herein. In some embodiments, the SC-β cell isgenetically modified to express neurogenin 3. In some embodiments,genetic modification of the SC-β cell comprise introducing a synthetic,modified mRNA encoding neurogenin 3. It is believed that geneticmodification of SC-β cells with synthetic, modified RNA encodingneurogenin 3 increases production of insulin form the cells. It isexpected that such genetic modification of any insulin producing cell isexpected to increased insulin production in that cell.

In some aspects, the disclosure provides a SC-β cell geneticallymodified to include a detectable marker at the insulin locus. In someembodiments, the SC-β cell is modified to replace both alleles of theinsulin locus with a detectable marker. In some embodiments, the SC-βcell is genetically modified to insert the detectable marker into theinsulin locus so that it is expressed with insulin in the SC-β cell inresponse to a glucose challenge. In some embodiments, the SC-β cell isgenetically modified to insert the detectable marker into the insulinlocus in place of insulin so that it is expressed instead of insulin inthe SC-β cell in response to a glucose challenge. It is contemplatedthat any detectable marker can be inserted into the insulin locus,including for example, a nucleic acid encoding a fluorescent protein(e.g., GFP). Those skilled in the art will appreciate that suchgenetically modified SC-β cells can be used in various screeningmethods, e.g., to identify agents which stimulate insulin expressionand/or secretion from β cells by assaying for the detectable marker inresponse to the agent. For example, an SC-β cell genetically modified toreplace the insulin gene at both alleles (e.g., with GFP) can becontacted with a test agent and those agents which cause the SC-β cellsto fluoresce due to expression of the GFP are considered to be candidateagents which are capable of activating insulin gene expression in βcells. In other words, the detectable marker may be used as a surrogatemarker for insulin expression in such genetically modified SC-β cells.

The term “identity” as used herein refers to the extent to which thesequence of two or more nucleic acids or polypeptides is the same. Thepercent identity between a sequence of interest and a second sequenceover a window of evaluation, e.g., over the length of the sequence ofinterest, may be computed by aligning the sequences, determining thenumber of residues (nucleotides or amino acids) within the window ofevaluation that are opposite an identical residue allowing theintroduction of gaps to maximize identity, dividing by the total numberof residues of the sequence of interest or the second sequence(whichever is greater) that fall within the window, and multiplying by100. When computing the number of identical residues needed to achieve aparticular percent identity, fractions are to be rounded to the nearestwhole number. Percent identity can be calculated with the use of avariety of computer programs known in the art. For example, computerprograms such as BLAST2, BLASTN, BLASTP, Gapped BLAST, etc., generatealignments and provide percent identity between sequences of interest.The algorithm of Karlin and Altschul (Karlin and Altschul, Proc. Natl.Acad. Sci. USA 87:22264-2268, 1990) modified as in Karlin and Altschul,Proc. Natl. Acad. ScL USA 90:5873-5877, 1993 is incorporated into theNBLAST and XBLAST programs of Altschul et al. (Altschul, et al., J. MoI.Biol. 215:403-410, 1990). To obtain gapped alignments for comparisonpurposes, Gapped BLAST is utilized as described in Altschul et al.(Altschul, et al. Nucleic Acids Res. 25: 3389-3402, 1997). Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs may be used. A PAM250 or BLOSUM62 matrix may beused. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information (NCBI). Seethe Web site having URL world-wide web address of: “ncbi.nlm nih.gov”for these programs. In a specific embodiment, percent identity iscalculated using BLAST2 with default parameters as provided by the NCBI.

The term “isolated” or “partially purified” as used herein refers, inthe case of a nucleic acid or polypeptide, to a nucleic acid orpolypeptide separated from at least one other component (e.g., nucleicacid or polypeptide) that is present with the nucleic acid orpolypeptide as found in its natural source and/or that would be presentwith the nucleic acid or polypeptide when expressed by a cell, orsecreted in the case of secreted polypeptides. A chemically synthesizednucleic acid or polypeptide or one synthesized using in vitrotranscription/translation is considered “isolated”.

The term “isolated cell” as used herein refers to a cell that has beenremoved from an organism in which it was originally found or adescendant of such a cell. Optionally the cell has been cultured invitro, e.g., in the presence of other cells. Optionally the cell islater introduced into a second organism or re-introduced into theorganism from which it (or the cell from which it is descended) wasisolated.

The term “isolated population” with respect to an isolated population ofcells as used herein refers to a population of cells that has beenremoved and separated from a mixed or heterogeneous population of cells.In some embodiments, an isolated population is a substantially purepopulation of cells as compared to the heterogeneous population fromwhich the cells were isolated or enriched from.

The term “substantially pure”, with respect to a particular cellpopulation, refers to a population of cells that is at least about 75%,preferably at least about 85%, more preferably at least about 90%, andmost preferably at least about 95% pure, with respect to the cellsmaking up a total cell population. Recast, the terms “substantiallypure” or “essentially purified”, with regard to a population of SC-βcells, refers to a population of cells that contain fewer than about20%, more preferably fewer than about 15%, 10%, 8%, 7%, most preferablyfewer than about 5%, 4%, 3%, 2%, 1%, or less than 1%, of cells that arenot SC-β cells as defined by the terms herein. In some embodiments, thepresent invention encompasses methods to expand a population of SC-βcells, wherein the expanded population of SC-β cells is a substantiallypure population of SC-β cells.

Similarly, with regard to a “substantially pure” or “essentiallypurified” population of insulin-positive endocrine cells refers to apopulation of cells that contain fewer than about 20%, more preferablyfewer than about 15%, 10%, 8%, 7%, most preferably fewer than about 5%,4%, 3%, 2%, 1%, or less than 1%, of cells that are not insulin-positiveendocrine cells as defined by the terms herein. In some embodiments, thepresent invention encompasses methods to expand a population ofinsulin-positive endocrine cells, wherein the expanded population ofinsulin-positive endocrine cells is a substantially pure population ofinsulin-positive endocrine cells.

Similarly, with regard to a “substantially pure” or “essentiallypurified” population of Ngn3-positive endocrine progenitors, refers to apopulation of cells that contain fewer than about 20%, more preferablyfewer than about 15%, 10%, 8%, 7%, most preferably fewer than about 5%,4%, 3%, 2%, 1%, or less than 1%, of cells that are not Ngn3-positiveendocrine progenitors or their progeny as defined by the terms herein.In some embodiments, the present invention encompasses methods to expanda population of Ngn3-positive endocrine progenitors, wherein theexpanded population of Ngn3-positive endocrine progenitors is asubstantially pure population of Ngn3-positive endocrine progenitors.

Similarly, with regard to a “substantially pure” or “essentiallypurified” population of Pdx1-positive, NKX6-1-positive pancreaticprogenitors, refers to a population of cells that contain fewer thanabout 20%, more preferably fewer than about 15%, 10%, 8%, 7%, mostpreferably fewer than about 5%, 4%, 3%, 2%, 1%, or less than 1%, ofcells that are not Pdx1-positive, NKX6-1-positive pancreatic progenitorsor their progeny as defined by the terms herein. In some embodiments,the present invention encompasses methods to expand a population ofPdx1-positive, NKX6-1-positive pancreatic progenitors, wherein theexpanded population of Pdx1-positive, NKX6-1-positive pancreaticprogenitors is a substantially pure population of Pdx1-positive,NKX6-1-positive pancreatic progenitors.

Similarly, with regard to a “substantially pure” or “essentiallypurified” population of Pdx1-positive pancreatic progenitors, refers toa population of cells that contain fewer than about 20%, more preferablyfewer than about 15%, 10%, 8%, 7%, most preferably fewer than about 5%,4%, 3%, 2%, 1%, or less than 1%, of cells that are not Pdx1-positivepancreatic progenitors or their progeny as defined by the terms herein.In some embodiments, the present invention encompasses methods to expanda population of Pdx1-positive pancreatic progenitors, wherein theexpanded population of Pdx1-positive pancreatic progenitors is asubstantially pure population of Pdx1-positive pancreatic progenitors.

Similarly, with regard to a “substantially pure” or “essentiallypurified” population of primitive gut tube cells, refers to a populationof cells that contain fewer than about 20%, more preferably fewer thanabout 15%, 10%, 8%, 7%, most preferably fewer than about 5%, 4%, 3%, 2%,1%, or less than 1%, of cells that are not primitive gut tube cells ortheir progeny as defined by the terms herein. In some embodiments, thepresent invention encompasses methods to expand a population ofprimitive gut tube cells, wherein the expanded population of primitivegut tube cells is a substantially pure population of primitive gut tubecells.

Similarly, with regard to a “substantially pure” or “essentiallypurified” population of definitive endoderm cells, refers to apopulation of cells that contain fewer than about 20%, more preferablyfewer than about 15%, 10%, 8%, 7%, most preferably fewer than about 5%,4%, 3%, 2%, 1%, or less than 1%, of cells that are not definitiveendoderm cells or their progeny as defined by the terms herein. In someembodiments, the present invention encompasses methods to expand apopulation of definitive endoderm cells, wherein the expanded populationof definitive endoderm cells is a substantially pure population ofdefinitive endoderm cells.

Similarly, with regard to a “substantially pure” or “essentiallypurified” population of pluripotent cells, refers to a population ofcells that contain fewer than about 20%, more preferably fewer thanabout 15%, 10%, 8%, 7%, most preferably fewer than about 5%, 4%, 3%, 2%,1%, or less than 1%, of cells that are not pluripotent cells or theirprogeny as defined by the terms herein. In some embodiments, the presentinvention encompasses methods to expand a population of pluripotentcells, wherein the expanded population of pluripotent cells is asubstantially pure population of pluripotent cells.

The terms “enriching” or “enriched” are used interchangeably herein andmean that the yield (fraction) of cells of one type is increased by atleast 10% over the fraction of cells of that type in the startingculture or preparation.

The terms “renewal” or “self-renewal” or “proliferation” are usedinterchangeably herein, are used to refer to the ability of stem cellsto renew themselves by dividing into the same non-specialized cell typeover long periods, and/or many months to years. In some instances,proliferation refers to the expansion of cells by the repeated divisionof single cells into two identical daughter cells.

The term “lineages” as used herein describes a cell with a commonancestry or cells with a common developmental fate. For example, in thecontext of a cell that is of endoderm origin or is “endodermal linage”this means the cell was derived from an endoderm cell and candifferentiate along the endoderm lineage restricted pathways, such asone or more developmental lineage pathways which give rise to definitiveendoderm cells, which in turn can differentiate into liver cells,thymus, pancreas, lung and intestine.

As used herein, the term “xenogeneic” refers to cells that are derivedfrom different species.

A “marker” as used herein is used to describe the characteristics and/orphenotype of a cell. Markers can be used for selection of cellscomprising characteristics of interests. Markers will vary with specificcells. Markers are characteristics, whether morphological, functional orbiochemical (enzymatic) characteristics of the cell of a particular celltype, or molecules expressed by the cell type. Preferably, such markersare proteins, and more preferably, possess an epitope for antibodies orother binding molecules available in the art. However, a marker mayconsist of any molecule found in a cell including, but not limited to,proteins (peptides and polypeptides), lipids, polysaccharides, nucleicacids and steroids. Examples of morphological characteristics or traitsinclude, but are not limited to, shape, size, and nuclear to cytoplasmicratio. Examples of functional characteristics or traits include, but arenot limited to, the ability to adhere to particular substrates, abilityto incorporate or exclude particular dyes, ability to migrate underparticular conditions, and the ability to differentiate along particularlineages. Markers may be detected by any method available to one ofskill in the art. Markers can also be the absence of a morphologicalcharacteristic or absence of proteins, lipids etc. Markers can be acombination of a panel of unique characteristics of the presence andabsence of polypeptides and other morphological characteristics.

The term “modulate” is used consistently with its use in the art, i.e.,meaning to cause or facilitate a qualitative or quantitative change,alteration, or modification in a process, pathway, or phenomenon ofinterest. Without limitation, such change may be an increase, decrease,or change in relative strength or activity of different components orbranches of the process, pathway, or phenomenon. A “modulator” is anagent that causes or facilitates a qualitative or quantitative change,alteration, or modification in a process, pathway, or phenomenon ofinterest.

As used herein, the term “DNA” is defined as deoxyribonucleic acid.

The term “polynucleotide” is used herein interchangeably with “nucleicacid” to indicate a polymer of nucleosides. Typically a polynucleotideof this invention is composed of nucleosides that are naturally found inDNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine,deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine)joined by phosphodiester bonds. However the term encompasses moleculescomprising nucleosides or nucleoside analogs containing chemically orbiologically modified bases, modified backbones, etc., whether or notfound in naturally occurring nucleic acids, and such molecules may bepreferred for certain applications. Where this application refers to apolynucleotide it is understood that both DNA, RNA, and in each caseboth single- and double-stranded forms (and complements of eachsingle-stranded molecule) are provided. “Polynucleotide sequence” asused herein can refer to the polynucleotide material itself and/or tothe sequence information (i.e. the succession of letters used asabbreviations for bases) that biochemically characterizes a specificnucleic acid. A polynucleotide sequence presented herein is presented ina 5′ to 3′ direction unless otherwise indicated.

The terms “polypeptide” as used herein refers to a polymer of aminoacids. The terms “protein” and “polypeptide” are used interchangeablyherein. A peptide is a relatively short polypeptide, typically betweenabout 2 and 60 amino acids in length. Polypeptides used herein typicallycontain amino acids such as the 20 L-amino acids that are most commonlyfound in proteins. However, other amino acids and/or amino acid analogsknown in the art can be used. One or more of the amino acids in apolypeptide may be modified, for example, by the addition of a chemicalentity such as a carbohydrate group, a phosphate group, a fatty acidgroup, a linker for conjugation, functionalization, etc. A polypeptidethat has a non-polypeptide moiety covalently or non-covalentlyassociated therewith is still considered a “polypeptide”. Exemplarymodifications include glycosylation and palmitoylation. Polypeptides maybe purified from natural sources, produced using recombinant DNAtechnology, synthesized through chemical means such as conventionalsolid phase peptide synthesis, etc. The term “polypeptide sequence” or“amino acid sequence” as used herein can refer to the polypeptidematerial itself and/or to the sequence information (i.e., the successionof letters or three letter codes used as abbreviations for amino acidnames) that biochemically characterizes a polypeptide. A polypeptidesequence presented herein is presented in an N-terminal to C-terminaldirection unless otherwise indicated.

The term a “variant” in referring to a polypeptide could be, e.g., apolypeptide at least 80%, 85%, 90%, 95%, 98%, or 99% identical to fulllength polypeptide. The variant could be a fragment of full lengthpolypeptide. The variant could be a naturally occurring splice variant.The variant could be a polypeptide at least 80%, 85%, 90%, 95%, 98%, or99% identical to a fragment of the polypeptide, wherein the fragment isat least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% as long as thefull length wild type polypeptide or a domain thereof having an activityof interest, such as the ability to detect the presence of a SC-β cell,or an insulin-positive endocrine cell or precursor thereof from whichthe SC-β cell is derived. In some embodiments the domain is at least100, 200, 300, or 400 amino acids in length, beginning at any amino acidposition in the sequence and extending toward the C-terminus. Variationsknown in the art to eliminate or substantially reduce the activity ofthe protein are preferably avoided. In some embodiments, the variantlacks an N- and/or C-terminal portion of the full length polypeptide,e.g., up to 10, 20, or 50 amino acids from either terminus is lacking.In some embodiments the polypeptide has the sequence of a mature (fulllength) polypeptide, by which is meant a polypeptide that has had one ormore portions such as a signal peptide removed during normalintracellular proteolytic processing (e.g., during co-translational orpost-translational processing). In some embodiments wherein the proteinis produced other than by purifying it from cells that naturally expressit, the protein is a chimeric polypeptide, by which is meant that itcontains portions from two or more different species. In someembodiments wherein a protein is produced other than by purifying itfrom cells that naturally express it, the protein is a derivative, bywhich is meant that the protein comprises additional sequences notrelated to the protein so long as those sequences do not substantiallyreduce the biological activity of the protein.

The term “functional fragments” as used herein is a polypeptide havingamino acid sequence which is smaller in size than, but substantiallyhomologous to the polypeptide it is a fragment of, and where thefunctional fragment polypeptide sequence is about at least 50%, or 60%or 70% or at 80% or 90% or 100% or greater than 100%, for example1.5-fold, 2-fold, 3-fold, 4-fold or greater than 4-fold effectivebiological action as the polypeptide from which it is a fragment of.Functional fragment polypeptides may have additional functions that caninclude decreased antigenicity, increased DNA binding (as intranscription factors), or altered RNA binding (as in regulating RNAstability or degradation).

The term “vector” refers to a carrier DNA molecule into which a DNAsequence can be inserted for introduction into a host cell. Preferredvectors are those capable of autonomous replication and/or expression ofnucleic acids to which they are linked. Vectors capable of directing theexpression of genes to which they are operatively linked are referred toherein as “expression vectors”. Thus, an “expression vector” is aspecialized vector that contains the necessary regulatory regions neededfor expression of a gene of interest in a host cell. In some embodimentsthe gene of interest is operably linked to another sequence in thevector. Vectors can be viral vectors or non-viral vectors. Should viralvectors be used, it is preferred the viral vectors are replicationdefective, which can be achieved for example by removing all viralnucleic acids that encode for replication. A replication defective viralvector will still retain its infective properties and enters the cellsin a similar manner as a replicating adenoviral vector, however onceadmitted to the cell a replication defective viral vector does notreproduce or multiply. Vectors also encompass liposomes andnanoparticles and other means to deliver DNA molecule to a cell.

The term “operably linked” means that the regulatory sequences necessaryfor expression of the coding sequence are placed in the DNA molecule inthe appropriate positions relative to the coding sequence so as toeffect expression of the coding sequence. This same definition issometimes applied to the arrangement of coding sequences andtranscription control elements (e.g. promoters, enhancers, andtermination elements) in an expression vector. The term “operativelylinked” includes having an appropriate start signal (e.g., ATG) in frontof the polynucleotide sequence to be expressed, and maintaining thecorrect reading frame to permit expression of the polynucleotidesequence under the control of the expression control sequence, andproduction of the desired polypeptide encoded by the polynucleotidesequence.

The term “viral vectors” refers to the use of viruses, orvirus-associated vectors as carriers of a nucleic acid construct into acell. Constructs may be integrated and packaged into non-replicating,defective viral genomes like Adenovirus, Adeno-associated virus (AAV),or Herpes simplex virus (HSV) or others, including reteroviral andlentiviral vectors, for infection or transduction into cells. The vectormay or may not be incorporated into the cell's genome. The constructsmay include viral sequences for transfection, if desired. Alternatively,the construct may be incorporated into vectors capable of episomalreplication, e.g EPV and EBV vectors.

The terms “regulatory sequence” and “promoter” are used interchangeablyherein, and refer to nucleic acid sequences, such as initiation signals,enhancers, and promoters, which induce or control transcription ofprotein coding sequences with which they are operatively linked. In someexamples, transcription of a recombinant gene is under the control of apromoter sequence (or other transcriptional regulatory sequence) whichcontrols the expression of the recombinant gene in a cell-type in whichexpression is intended. It will also be understood that the recombinantgene can be under the control of transcriptional regulatory sequenceswhich are the same or which are different from those sequences whichcontrol transcription of the naturally-occurring form of a protein. Insome instances the promoter sequence is recognized by the syntheticmachinery of the cell, or introduced synthetic machinery, required forinitiating transcription of a specific gene.

As used herein, the term “transcription factor” refers to a protein thatbinds to specific parts of DNA using DNA binding domains and is part ofthe system that controls the transfer (or transcription) of geneticinformation from DNA to RNA. As used herein, “proliferating” and“proliferation” refer to an increase in the number of cells in apopulation (growth) by means of cell division. Cell proliferation isgenerally understood to result from the coordinated activation ofmultiple signal transduction pathways in response to the environment,including growth factors and other mitogens. Cell proliferation may alsobe promoted by release from the actions of intra- or extracellularsignals and mechanisms that block or negatively affect cellproliferation.

The term “selectable marker” refers to a gene, RNA, or protein that whenexpressed, confers upon cells a selectable phenotype, such as resistanceto a cytotoxic or cytostatic agent (e.g., antibiotic resistance),nutritional prototrophy, or expression of a particular protein that canbe used as a basis to distinguish cells that express the protein fromcells that do not. Proteins whose expression can be readily detectedsuch as a fluorescent or luminescent protein or an enzyme that acts on asubstrate to produce a colored, fluorescent, or luminescent substance(“detectable markers”) constitute a subset of selectable markers. Thepresence of a selectable marker linked to expression control elementsnative to a gene that is normally expressed selectively or exclusivelyin pluripotent cells makes it possible to identify and select somaticcells that have been reprogrammed to a pluripotent state. A variety ofselectable marker genes can be used, such as neomycin resistance gene(neo), puromycin resistance gene (puro), guanine phosphoribosyltransferase (gpt), dihydrofolate reductase (DHFR), adenosine deaminase(ada), puromycin-N-acetyltransferase (PAC), hygromycin resistance gene(hyg), multidrug resistance gene (mdr), thymidine kinase (TK),hypoxanthine-guanine phosphoribosyltransferase (HPRT), and hisD gene.Detectable markers include green fluorescent protein (GFP) blue,sapphire, yellow, red, orange, and cyan fluorescent proteins andvariants of any of these. Luminescent proteins such as luciferase (e.g.,firefly or Renilla luciferase) are also of use. As will be evident toone of skill in the art, the term “selectable marker” as used herein canrefer to a gene or to an expression product of the gene, e.g., anencoded protein.

In some embodiments the selectable marker confers a proliferation and/orsurvival advantage on cells that express it relative to cells that donot express it or that express it at significantly lower levels. Suchproliferation and/or survival advantage typically occurs when the cellsare maintained under certain conditions, i.e., “selective conditions.”To ensure an effective selection, a population of cells can bemaintained for a under conditions and for a sufficient period of timesuch that cells that do not express the marker do not proliferate and/ordo not survive and are eliminated from the population or their number isreduced to only a very small fraction of the population. The process ofselecting cells that express a marker that confers a proliferationand/or survival advantage by maintaining a population of cells underselective conditions so as to largely or completely eliminate cells thatdo not express the marker is referred to herein as “positive selection”,and the marker is said to be “useful for positive selection”. Negativeselection and markers useful for negative selection are also of interestin certain of the methods described herein. Expression of such markersconfers a proliferation and/or survival disadvantage on cells thatexpress the marker relative to cells that do not express the marker orexpress it at significantly lower levels (or, considered another way,cells that do not express the marker have a proliferation and/orsurvival advantage relative to cells that express the marker). Cellsthat express the marker can therefore be largely or completelyeliminated from a population of cells when maintained in selectiveconditions for a sufficient period of time.

A “reporter gene” as used herein encompasses any gene that isgenetically introduced into a cell that adds to the phenotype of thestem cell. Reporter genes as disclosed in this invention are intended toencompass fluorescent, luminescent, enzymatic and resistance genes, butalso other genes which can easily be detected by persons of ordinaryskill in the art. In some embodiments of the invention, reporter genesare used as markers for the identification of particular stem cells,cardiovascular stem cells and their differentiated progeny. A reportergene is generally operatively linked to sequences that regulate itsexpression in a manner dependent upon one or more conditions which aremonitored by measuring expression of the reporter gene. In some cases,expression of the reporter gene may be determined in live cells. Wherelive cell reporter gene assays are used, reporter gene expression may bemonitored at multiple time points, e.g., 2, 3, 4, 5, 6, 8, or 10 or moretime points. In some cases, where a live cell reporter assay is used,reporter gene expression is monitored with a frequency of at least about10 minutes to about 24 hours, e.g., 20 minutes, 1 hour, 2 hours, 3hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours,12 hours, 18 hours, or another frequency from any integer between about10 minutes to about 24 hours.

The terms “subject” and “individual” are used interchangeably herein,and refer to an animal, for example, a human from whom cells can beobtained and/or to whom treatment, including prophylactic treatment,with the cells as described herein, is provided. For treatment of thoseinfections, conditions or disease states which are specific for aspecific animal such as a human subject, the term subject refers to thatspecific animal. The “non-human animals” and “non-human mammals” as usedinterchangeably herein, includes mammals such as rats, mice, rabbits,sheep, cats, dogs, cows, pigs, and non-human primates. The term“subject” also encompasses any vertebrate including but not limited tomammals, reptiles, amphibians and fish. However, advantageously, thesubject is a mammal such as a human, or other mammals such as adomesticated mammal, e.g. dog, cat, horse, and the like, or productionmammal, e.g. cow, sheep, pig, and the like.

The terms “diabetes” and “diabetes mellitus” are used interchangeablyherein. The World Health Organization defines the diagnostic value offasting plasma glucose concentration to 7.0 mmol/l (126 mg/dl) and abovefor Diabetes Mellitus (whole blood 6.1 mmol/l or 110 mg/dl), or 2-hourglucose level 11.1 mmol/L or higher (200 mg/dL or higher). Other valuessuggestive of or indicating high risk for Diabetes Mellitus includeelevated arterial pressure 140/90 mm Hg or higher; elevated plasmatriglycerides (1.7 mmol/L; 150 mg/dL) and/or low HDL-cholesterol (lessthan 0.9 mmol/L, 35 mg/dl for men; less than 1.0 mmol/L, 39 mg/dLwomen); central obesity (males: waist to hip ratio higher than 0.90;females: waist to hip ratio higher than 0.85) and/or body mass indexexceeding 30 kg/m²; microalbuminuria, where the urinary albuminexcretion rate 20 μg/min or higher, or albumin:creatinine ratio 30 mg/gor higher). The term diabetes encompasses all forms of diabetes, e.g.Type I, Type II and Type 1.5.

The terms “treat”, “treating”, “treatment”, etc., as applied to anisolated cell, include subjecting the cell to any kind of process orcondition or performing any kind of manipulation or procedure on thecell. As applied to a subject, the terms refer to providing medical orsurgical attention, care, or management to an individual. The individualis usually ill or injured, or at increased risk of becoming ill relativeto an average member of the population and in need of such attention,care, or management.

As used herein, the term “treating” and “treatment” refers toadministering to a subject an effective amount of a composition so thatthe subject as a reduction in at least one symptom of the disease or animprovement in the disease, for example, beneficial or desired clinicalresults. For purposes of this invention, beneficial or desired clinicalresults include, but are not limited to, alleviation of one or moresymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. Treating canrefer to prolonging survival as compared to expected survival if notreceiving treatment. Thus, one of skill in the art realizes that atreatment may improve the disease condition, but may not be a completecure for the disease. As used herein, the term “treatment” includesprophylaxis. Alternatively, treatment is “effective” if the progressionof a disease is reduced or halted. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.Those in need of treatment include those already diagnosed with acardiac condition, as well as those likely to develop a cardiaccondition due to genetic susceptibility or other factors such as weight,diet and health.

As used herein, the terms “administering,” “introducing” and“transplanting” are used interchangeably in the context of the placementof cells, e.g., SC-β cells) of the invention into a subject, by a methodor route which results in at least partial localization of theintroduced cells at a desired site. The cells e.g. SC-β cells (e.g.,pancreatic β cells or pancreatic β-like cells) can be implanted directlyto the pancreas, or alternatively be administered by any appropriateroute which results in delivery to a desired location in the subjectwhere at least a portion of the implanted cells or components of thecells remain viable. The period of viability of the cells afteradministration to a subject can be as short as a few hours, e.g.twenty-four hours, to a few days, to as long as several years. In someinstances, the cells can also be administered at a non-pancreaticlocation, such as in the liver or subcutaneously, for example, in acapsule (e.g., microcapsule) to maintain the implanted cells at theimplant location and avoid migration of the implanted cells.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intraventricular, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, sub capsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. The phrases “systemicadministration,” “administered systemically”, “peripheraladministration” and “administered peripherally” as used herein mean theadministration of cardiovascular stem cells and/or their progeny and/orcompound and/or other material other than directly into the centralnervous system, such that it enters the animal's system and, thus, issubject to metabolism and other like processes, for example,subcutaneous administration.

The term “tissue” refers to a group or layer of specialized cells whichtogether perform certain special functions. The term “tissue-specific”refers to a source of cells from a specific tissue.

The terms “decrease”, “reduced”, “reduction”, “decrease” or “inhibit”are all used herein generally to mean a decrease by a statisticallysignificant amount. However, for avoidance of doubt, “reduced”,“reduction” or “decrease” or “inhibit” means a decrease by at least 10%as compared to a reference level, for example a decrease by at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% decrease(i.e. absent level as compared to a reference sample), or any decreasebetween 10-100% as compared to a reference level.

The terms “increased”, “increase” or “enhance” or “activate” are allused herein to generally mean an increase by a statically significantamount; for the avoidance of any doubt, the terms “increased”,“increase” or “enhance” or “activate” means an increase of at least 10%as compared to a reference level, for example an increase of at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% increaseor any increase between 10-100% as compared to a reference level, or atleast about a 2-fold, or at least about a 3-fold, or at least about a4-fold, or at least about a 5-fold or at least about a 10-fold increase,or any increase between 2-fold and 10-fold or greater as compared to areference level.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) below normal, or lower, concentration of the marker. The termrefers to statistical evidence that there is a difference. It is definedas the probability of making a decision to reject the null hypothesiswhen the null hypothesis is actually true. The decision is often madeusing the p-value.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, references to “the method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

Stem Cells

Stem cells are cells that retain the ability to renew themselves throughmitotic cell division and can differentiate into a diverse range ofspecialized cell types. The two broad types of mammalian stem cells are:embryonic stem (ES) cells that are found in blastocysts, and adult stemcells that are found in adult tissues. In a developing embryo, stemcells can differentiate into all of the specialized embryonic tissues.In adult organisms, stem cells and progenitor cells act as a repairsystem for the body, replenishing specialized cells, but also maintainthe normal turnover of regenerative organs, such as blood, skin orintestinal tissues. Pluripotent stem cells can differentiate into cellsderived from any of the three germ layers.

While certain embodiments are described below in reference to the use ofstem cells for producing SC-β cells (e.g., mature pancreatic β cells orβ-like cells) or precursors thereof, germ cells may be used in place of,or with, the stem cells to provide at least one SC-β cell, using similarprotocols as the illustrative protocols described herein. Suitable germcells can be prepared, for example, from primordial germ cells presentin human fetal material taken about 8-11 weeks after the last menstrualperiod. Illustrative germ cell preparation methods are described, forexample, in Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998and U.S. Pat. No. 6,090,622.

ES cells, e.g., human embryonic stem cells (hESCs) or mouse embryonicstem cells (mESCs), with a virtually endless replication capacity andthe potential to differentiate into most cell types, present, inprinciple, an unlimited starting material to generate the differentiatedcells for clinical therapy (worldwide web at subdomainstemcells.nih.gov/info/scireport/2006report.htm). One possibleapplication of ES cells is to generate new pancreatic β cells for thecell replacement therapy of type I diabetics, by first producingendoderm, e.g., definitive endoderm, from, e.g., hESCs, and then furtherdifferentiating the definitive endoderm into at least oneinsulin-positive endocrine cell or precursor thereof, and then furtherdifferentiating the at least one insulin-positive endocrine cell orprecursor thereof into a SC-β cell.

hESC cells, are described, for example, by Cowan et al. (N Engl. J. Med.350:1353, 2004) and Thomson et al. (Science 282:1145, 1998); embryonicstem cells from other primates, Rhesus stem cells (Thomson et al., Proc.Natl. Acad. Sci. USA 92:7844, 1995), marmoset stem cells (Thomson etal., Biol. Reprod. 55:254, 1996) and human embryonic germ (hEG) cells(Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998) may alsobe used in the methods disclosed herein. mESCs, are described, forexample, by Tremml et al. (Curr Protoc Stem Cell Biol. Chapter 1:Unit1C.4, 2008). The stem cells may be, for example, unipotent, totipotent,multipotent, or pluripotent. In some examples, any cells of primateorigin that are capable of producing progeny that are derivatives of atleast one germinal layer, or all three germinal layers, may be used inthe methods disclosed herein.

In certain examples, ES cells may be isolated, for example, as describedin Cowan et al. (N Engl. J. Med. 350:1353, 2004) and U.S. Pat. No.5,843,780 and Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995.For example, hESCs cells can be prepared from human blastocyst cellsusing the techniques described by Thomson et al. (U.S. Pat. No.6,200,806; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133 ff.,1998) and Reubinoff et al, Nature Biotech. 18:399, 2000. Equivalent celltypes to hESCs include their pluripotent derivatives, such as primitiveectoderm-like (EPL) cells, as outlined, for example, in WO 01/51610(Bresagen). hESCs can also be obtained from human pre-implantationembryos. Alternatively, in vitro fertilized (IVF) embryos can be used,or one-cell human embryos can be expanded to the blastocyst stage(Bongso et al., Hum Reprod 4: 706, 1989). Embryos are cultured to theblastocyst stage in G1.2 and G2.2 medium (Gardner et al., Fertil.Steril. 69:84, 1998). The zona pellucida is removed from developedblastocysts by brief exposure to pronase (Sigma). The inner cell massescan be isolated by immunosurgery, in which blastocysts are exposed to a1:50 dilution of rabbit anti-human spleen cell antiserum for 30 min,then washed for 5 min three times in DMEM, and exposed to a 1:5 dilutionof Guinea pig complement (Gibco) for 3 min (Solter et al., Proc. Natl.Acad. Sci. USA 72:5099, 1975). After two further washes in DMEM, lysedtrophectoderm cells are removed from the intact inner cell mass (ICM) bygentle pipetting, and the ICM plated on mEF feeder layers. After 9 to 15days, inner cell mass-derived outgrowths can be dissociated into clumps,either by exposure to calcium and magnesium-free phosphate-bufferedsaline (PBS) with 1 mM EDTA, by exposure to dispase or trypsin, or bymechanical dissociation with a micropipette; and then replated on mEF infresh medium. Growing colonies having undifferentiated morphology can beindividually selected by micropipette, mechanically dissociated intoclumps, and replated. ES-like morphology is characterized as compactcolonies with apparently high nucleus to cytoplasm ratio and prominentnucleoli. Resulting hESCs can then be routinely split every 1-2 weeks,for example, by brief trypsinization, exposure to Dulbecco's PBS(containing 2 mM EDTA), exposure to type IV collagenase (about 200 U/mL;Gibco) or by selection of individual colonies by micropipette. In someexamples, clump sizes of about 50 to 100 cells are optimal. mESCs cellscan be prepared from using the techniques described by e.g., Conner etal. (Curr. Prot. in Mol. Biol. Unit 23.4, 2003).

Embryonic stem cells can be isolated from blastocysts of members of theprimate species (U.S. Pat. No. 5,843,780; Thomson et al., Proc. Natl.Acad. Sci. USA 92:7844, 1995). Human embryonic stem (hES) cells can beprepared from human blastocyst cells using the techniques described byThomson et al. (U.S. Pat. No. 6,200,806; Science 282:1145, 1998; Curr.Top. Dev. Biol. 38:133 ff., 1998) and Reubinoff et al, Nature Biotech.18:399, 2000. Equivalent cell types to hES cells include theirpluripotent derivatives, such as primitive ectoderm-like (EPL) cells, asoutlined in WO 01/51610 (Bresagen).

Alternatively, in some embodiments, hES cells can be obtained from humanpreimplantation embryos. Alternatively, in vitro fertilized (IVF)embryos can be used, or one-cell human embryos can be expanded to theblastocyst stage (Bongso et al., Hum Reprod 4: 706, 1989). Embryos arecultured to the blastocyst stage in G1.2 and G2.2 medium (Gardner etal., Fertil. Steril. 69:84, 1998). The zona pellucida is removed fromdeveloped blastocysts by brief exposure to pronase (Sigma). The innercell masses are isolated by immunosurgery, in which blastocysts areexposed to a 1:50 dilution of rabbit anti-human spleen cell antiserumfor 30 min, then washed for 5 min three times in DMEM, and exposed to a1:5 dilution of Guinea pig complement (Gibco) for 3 min (Solter et al.,Proc. Natl. Acad. Sci. USA 72:5099, 1975). After two further washes inDMEM, lysed trophectoderm cells are removed from the intact inner cellmass (ICM) by gentle pipetting, and the ICM plated on mEF feeder layers.

After 9 to 15 days, inner cell mass-derived outgrowths are dissociatedinto clumps, either by exposure to calcium and magnesium-freephosphate-buffered saline (PBS) with 1 mM EDTA, by exposure to dispaseor trypsin, or by mechanical dissociation with a micropipette; and thenreplated on mEF in fresh medium. Growing colonies havingundifferentiated morphology are individually selected by micropipette,mechanically dissociated into clumps, and replated. ES-like morphologyis characterized as compact colonies with apparently high nucleus tocytoplasm ratio and prominent nucleoli. Resulting ES cells are thenroutinely split every 1-2 weeks by brief trypsinization, exposure toDulbecco's PBS (containing 2 mM EDTA), exposure to type IV collagenase(^(˜)200 U/mL; Gibco) or by selection of individual colonies bymicropipette. Clump sizes of about 50 to 100 cells are optimal.

In some embodiments, human Embryonic Germ (hEG) cells are pluripotentstem cells which can be used in the methods as disclosed herein todifferentiate into primitive endoderm cells. hEG cells can be used beprepared from primordial germ cells present in human fetal materialtaken about 8-11 weeks after the last menstrual period. Suitablepreparation methods are described in Shamblott et al., Proc. Natl. Acad.Sci. USA 95:13726, 1998 and U.S. Pat. No. 6,090,622, which isincorporated herein in its entirety by reference.

Briefly, genital ridges processed to form disaggregated cells. EG growthmedium is DMEM, 4500 mg/L D-glucose, 2200 mg/L mM NaHCO₃; 15% ESqualified fetal calf serum (BRL); 2 mM glutamine (BRL); 1 mM sodiumpyruvate (BRL); 1000-2000 U/mL human recombinant leukemia inhibitoryfactor (LIF, Genzyme); 1-2 ng/mL human recombinant bFGF (Genzyme); and 1μM forskolin (in 10% DMSO). Ninety-six well tissue culture plates areprepared with a sub-confluent layer of feeder cells (e.g., STO cells,ATCC No. CRL 1503) cultured for 3 days in modified EG growth medium freeof LIF, bFGF or forskolin, inactivated with 5000 rad γ-irradiation^(˜)0.2 mL of primary germ cell (PGC) suspension is added to each of thewells. The first passage is done after 7-10 days in EG growth medium,transferring each well to one well of a 24-well culture dish previouslyprepared with irradiated STO mouse fibroblasts. The cells are culturedwith daily replacement of medium until cell morphology consistent withEG cells is observed, typically after 7-30 days or 1-4 passages.

In certain examples, the stem cells can be undifferentiated (e.g. a cellnot committed to a specific linage) prior to exposure to at least one βcell maturation factor according to the methods as disclosed herein,whereas in other examples it may be desirable to differentiate the stemcells to one or more intermediate cell types prior to exposure of the atleast one β cell maturation factor (s) described herein. For example,the stems cells may display morphological, biological or physicalcharacteristics of undifferentiated cells that can be used todistinguish them from differentiated cells of embryo or adult origin. Insome examples, undifferentiated cells may appear in the two dimensionsof a microscopic view in colonies of cells with high nuclear/cytoplasmicratios and prominent nucleoli. The stem cells may be themselves (forexample, without substantially any undifferentiated cells being present)or may be used in the presence of differentiated cells. In certainexamples, the stem cells may be cultured in the presence of suitablenutrients and optionally other cells such that the stem cells can growand optionally differentiate. For example, embryonic fibroblasts orfibroblast-like cells may be present in the culture to assist in thegrowth of the stem cells. The fibroblast may be present during one stageof stem cell growth but not necessarily at all stages. For example, thefibroblast may be added to stem cell cultures in a first culturing stageand not added to the stem cell cultures in one or more subsequentculturing stages.

Stem cells used in all aspects of the present invention can be any cellsderived from any kind of tissue (for example embryonic tissue such asfetal or pre-fetal tissue, or adult tissue), which stem cells have thecharacteristic of being capable under appropriate conditions ofproducing progeny of different cell types, e.g. derivatives of all of atleast one of the 3 germinal layers (endoderm, mesoderm, and ectoderm).These cell types may be provided in the form of an established cellline, or they may be obtained directly from primary embryonic tissue andused immediately for differentiation. Included are cells listed in theNIH Human Embryonic Stem Cell Registry, e.g. hESBGN-01, hESBGN-02,hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4,HES-5, HES-6 (ES Cell International); Miz-hES1 (MizMedi Hospital-SeoulNational University); HSF-1, HSF-6 (University of California at SanFrancisco); and H1, H7, H9, H13, H14 (Wisconsin Alumni ResearchFoundation (WiCell Research Institute)). In some embodiments, the sourceof human stem cells or pluripotent stem cells used forchemically-induced differentiation into mature, insulin positive cellsdid not involve destroying a human embryo.

In another embodiment, the stem cells can be isolated from tissueincluding solid tissue. In some embodiments, the tissue is skin, fattissue (e.g. adipose tissue), muscle tissue, heart or cardiac tissue. Inother embodiments, the tissue is for example but not limited to,umbilical cord blood, placenta, bone marrow, or chondral.

Stem cells of interest also include embryonic cells of various types,exemplified by human embryonic stem (hES) cells, described by Thomson etal. (1998) Science 282:1145; embryonic stem cells from other primates,such as Rhesus stem cells (Thomson et al. (1995) Proc. Natl. Acad. Sci.USA 92:7844); marmoset stem cells (Thomson et al. (1996) Biol. Reprod.55:254); and human embryonic germ (hEG) cells (Shambloft et al., Proc.Natl. Acad. Sci. USA 95:13726, 1998). Also of interest are lineagecommitted stem cells, such as mesodermal stem cells and other earlycardiogenic cells (see Reyes et al. (2001) Blood 98:2615-2625; Eisenberg& Bader (1996) Circ Res. 78(2):205-16; etc.) The stem cells may beobtained from any mammalian species, e.g. human, equine, bovine,porcine, canine, feline, rodent, e.g. mice, rats, hamster, primate, etc.In some embodiments, a human embryo was not destroyed for the source ofpluripotent cell used on the methods and compositions as disclosedherein.

ES cells are considered to be undifferentiated when they have notcommitted to a specific differentiation lineage. Such cells displaymorphological characteristics that distinguish them from differentiatedcells of embryo or adult origin. Undifferentiated ES cells are easilyrecognized by those skilled in the art, and typically appear in the twodimensions of a microscopic view in colonies of cells with highnuclear/cytoplasmic ratios and prominent nucleoli. Undifferentiated EScells express genes that may be used as markers to detect the presenceof undifferentiated cells, and whose polypeptide products may be used asmarkers for negative selection. For example, see U.S. application Ser.No. 2003/0224411 A1; Bhattacharya (2004) Blood 103(8):2956-64; andThomson (1998), supra., each herein incorporated by reference. Human EScell lines express cell surface markers that characterizeundifferentiated nonhuman primate ES and human EC cells, includingstage-specific embryonic antigen (SSEA)-3, SSEA-4, TRA-1-60, TRA-1-81,and alkaline phosphatase. The globo-series glycolipid GL7, which carriesthe SSEA-4 epitope, is formed by the addition of sialic acid to theglobo-series glycolipid GbS, which carries the SSEA-3 epitope. Thus, GL7reacts with antibodies to both SSEA-3 and SSEA-4. The undifferentiatedhuman ES cell lines did not stain for SSEA-1, but differentiated cellsstained strongly for SSEA-I. Methods for proliferating hES cells in theundifferentiated form are described in WO 99/20741, WO 01/51616, and WO03/020920.

A mixture of cells from a suitable source of endothelial, muscle, and/orneural stem cells can be harvested from a mammalian donor by methodsknown in the art. A suitable source is the hematopoieticmicroenvironment. For example, circulating peripheral blood, preferablymobilized (i.e., recruited), may be removed from a subject.Alternatively, bone marrow may be obtained from a mammal, such as ahuman patient, undergoing an autologous transplant. In some embodiments,stem cells can be obtained from the subjects adipose tissue, for exampleusing the CELUTION™ SYSTEM from Cytori, as disclosed in U.S. Pat. Nos.7,390,484 and 7,429,488 which is incorporated herein in its entirety byreference.

In some embodiments, human umbilical cord blood cells (HUCBC) are usefulin the methods as disclosed herein. Human UBC cells are recognized as arich source of hematopoietic and mesenchymal progenitor cells (Broxmeyeret al., 1992 Proc. Natl. Acad. Sci. USA 89:4109-4113). Previously,umbilical cord and placental blood were considered a waste productnormally discarded at the birth of an infant. Cord blood cells are usedas a source of transplantable stem and progenitor cells and as a sourceof marrow repopulating cells for the treatment of malignant diseases(i.e. acute lymphoid leukemia, acute myeloid leukemia, chronic myeloidleukemia, myelodysplastic syndrome, and nueroblastoma) and non-malignantdiseases such as Fanconi's anemia and aplastic anemia (Kohli-Kumar etal., 1993 Br. J. Haematol. 85:419-422; Wagner et al., 1992 Blood 79;1874-1881; Lu et al., 1996 Crit. Rev. Oncol. Hematol 22:61-78; Lu etal., 1995 Cell Transplantation 4:493-503). A distinct advantage of HUCBCis the immature immunity of these cells that is very similar to fetalcells, which significantly reduces the risk for rejection by the host(Taylor & Bryson, 1985 J. Immunol. 134:1493-1497). Human umbilical cordblood contains mesenchymal and hematopoietic progenitor cells, andendothelial cell precursors that can be expanded in tissue culture(Broxmeyer et al., 1992 Proc. Natl. Acad. Sci. USA 89:4109-4113;Kohli-Kumar et al., 1993 Br. J. Haematol. 85:419-422; Wagner et al.,1992 Blood 79; 1874-1881; Lu et al., 1996 Crit. Rev. Oncol. Hematol22:61-78; Lu et al., 1995 Cell Transplantation 4:493-503; Taylor &Bryson, 1985J. Immunol. 134:1493-1497 Broxmeyer, 1995 Transfusion35:694-702; Chen et al., 2001 Stroke 32:2682-2688; Nieda et al., 1997Br. J. Haematology 98:775-777; Erices et al., 2000 Br. J. Haematology109:235-242). The total content of hematopoietic progenitor cells inumbilical cord blood equals or exceeds bone marrow, and in addition, thehighly proliferative hematopoietic cells are eightfold higher in HUCBCthan in bone marrow and express hematopoietic markers such as CD14,CD34, and CD45 (Sanchez-Ramos et al., 2001 Exp. Neur. 171:109-115;Bicknese et al., 2002 Cell Transplantation 11:261-264; Lu et al., 1993J. Exp Med. 178:2089-2096).

In another embodiment, pluripotent cells are cells in the hematopoieticmicro-environment, such as the circulating peripheral blood, preferablyfrom the mononuclear fraction of peripheral blood, umbilical cord blood,bone marrow, fetal liver, or yolk sac of a mammal. The stem cells,especially neural stem cells, may also be derived from the centralnervous system, including the meninges.

In another embodiment, pluripotent cells are present in embryoid bodiesare formed by harvesting ES cells with brief protease digestion, andallowing small clumps of undifferentiated human ESCs to grow insuspension culture. Differentiation is induced by withdrawal ofconditioned medium. The resulting embryoid bodies are plated ontosemi-solid substrates. Formation of differentiated cells may be observedafter around about 7 days to around about 4 weeks. Viabledifferentiating cells from in vitro cultures of stem cells are selectedfor by partially dissociating embryoid bodies or similar structures toprovide cell aggregates. Aggregates comprising cells of interest areselected for phenotypic features using methods that substantiallymaintain the cell to cell contacts in the aggregate.

In an alternative embodiment, the stem cells can be reprogrammed stemcells, such as stem cells derived from somatic or differentiated cells.In such an embodiment, the de-differentiated stem cells can be forexample, but not limited to, neoplastic cells, tumor cells and cancercells or alternatively induced reprogrammed cells such as inducedpluripotent stem cells or iPS cells.

Cloning and Cell Culture

Illustrative methods for molecular genetics and genetic engineering thatmay be used in the technology described herein may be found, forexample, in current editions of Molecular Cloning: A Laboratory Manual,(Sambrook et al., Cold Spring Harbor); Gene Transfer Vectors forMammalian Cells (Miller & Calos eds.); and Current Protocols inMolecular Biology (F. M. Ausubel et al. eds., Wiley & Sons). Cellbiology, protein chemistry, and antibody techniques can be found, forexample, in Current Protocols in Protein Science (J. E. Colligan et al.eds., Wiley & Sons); Current Protocols in Cell Biology (J. S. Bonifacinoet al., Wiley & Sons) and Current protocols in Immunology (J. E.Colligan et al. eds., Wiley & Sons.). Illustrative reagents, cloningvectors, and kits for genetic manipulation may be commercially obtained,for example, from BioRad, Stratagene, Invitrogen, ClonTech, andSigma-Aldrich Co.

Suitable cell culture methods may be found, for example, in Cell culturemethods are described generally in the current edition of Culture ofAnimal Cells: A Manual of Basic Technique (R. I. Freshney ed., Wiley &Sons); General Techniques of Cell Culture (M. A. Harrison & I. F. Rae,Cambridge Univ. Press), and Embryonic Stem Cells: Methods and Protocols(K. Turksen ed., Humana Press). Suitable tissue culture supplies andreagents are commercially available, for example, from Gibco/BRL,Nalgene-Nunc International, Sigma Chemical Co., and ICN Biomedicals.

Pluripotent stem cells can be propagated by one of ordinary skill in theart and continuously in culture, using culture conditions that promoteproliferation without promoting differentiation. Exemplaryserum-containing ES medium is made with 80% DMEM (such as Knock-OutDMEM, Gibco), 20% of either defined fetal bovine serum (FBS, Hyclone) orserum replacement (WO 98/30679), 1% non-essential amino acids, 1 mML-glutamine, and 0.1 mM β-mercaptoethanol. Just before use, human bFGFis added to 4 ng/mL (WO 99/20741, Geron Corp.). Traditionally, ES cellsare cultured on a layer of feeder cells, typically fibroblasts derivedfrom embryonic or fetal tissue.

Scientists at Geron have discovered that pluripotent SCs can bemaintained in an undifferentiated state even without feeder cells. Theenvironment for feeder-free cultures includes a suitable culturesubstrate, particularly an extracellular matrix such as MATRIGEL™ orlaminin. Typically, enzymatic digestion is halted before cells becomecompletely dispersed (about 0.5 min with collagenase IV). Clumps of^(˜)10 to 2,000 cells are then plated directly onto the substratewithout further dispersal.

Feeder-free cultures are supported by a nutrient medium containingfactors that support proliferation of the cells without differentiation.Such factors may be introduced into the medium by culturing the mediumwith cells secreting such factors, such as irradiated (^(˜)4,000 rad)primary mouse embryonic fibroblasts, telomerized mouse fibroblasts, orfibroblast-like cells derived from pPS cells. Medium can be conditionedby plating the feeders at a density of ^(˜)5-6×10⁴ cm⁻² in a serum freemedium such as KO DMEM supplemented with 20% serum replacement and 4ng/mL bFGF. Medium that has been conditioned for 1-2 days issupplemented with further bFGF, and used to support pluripotent SCculture for 1-2 days. Features of the feeder-free culture method arefurther discussed in International Patent Publication WO 01/51616; andXu et al., Nat. Biotechnol. 19:971, 2001.

Under the microscope, ES cells appear with high nuclear/cytoplasmicratios, prominent nucleoli, and compact colony formation with poorlydiscernable cell junctions. Primate ES cells express stage-specificembryonic antigens (SSEA) 3 and 4, and markers detectable usingantibodies designated Tra-1-60 and Tra-1-81 (Thomson et al., Science282:1145, 1998). Mouse ES cells can be used as a positive control forSSEA-1, and as a negative control for SSEA-4, Tra-1-60, and Tra-1-81.SSEA-4 is consistently present human embryonal carcinoma (hEC) cells.Differentiation of pluripotent SCs in vitro results in the loss ofSSEA-4, Tra-1-60, and Tra-1-81 expression, and increased expression ofSSEA-1, which is also found on undifferentiated hEG cells.

Stem Cell-Derived β cell (SC-β)

In some aspects, the disclosure provides a stem cell-derived β cell(SC-β). The SC-β cells disclosed herein share many distinguishingfeatures of native β cells, but are different in certain aspects (e.g.,gene expression profiles). In some embodiments, the SC-β cell isnon-native. As used herein, “non-native” means that the SC-β cells aremarkedly different in certain aspects from β cells which exist innature, i.e., native § cells. It should be appreciated, however, thatthese marked differences typically pertain to structural features whichmay result in the SC-β cells exhibiting certain functional differences,e.g., although the gene expression patterns of SC-β cells differs fromnative β cells, the SC-β cells behave in a similar manner to native βcells but certain functions may be altered (e.g., improved) compared tonative β cells. For example, as is shown in FIG. 2E, a higher frequencyof SC-β cells respond to 20 mM glucose compared to the frequency ofnative β cells. Other differences between SC-β cells and native β cellswould be apparent to the skilled artisan based on the data disclosedherein.

The SC-β cells of the disclosure share many characteristic features of βcells which are important for normal β cell function. In someembodiments, the SC-β cell exhibits a glucose stimulated insulinsecretion (GSIS) response in vitro. In some embodiments, the SC-β cellexhibits a GSIS response in vivo. In some embodiments, the SC-β cellexhibits in vitro and in vivo GSIS responses. In some embodiments, theGSIS responses resemble the GSIS responses of an endogenous maturepancreatic β cell. In some embodiments, the SC-β cell exhibits a GSISresponse to at least one glucose challenge. In some embodiments, theSC-β cell exhibits a GSIS response to at least two sequential glucosechallenges. In some embodiments, the SC-β cell exhibits a GSIS responseto at least three sequential glucose challenges. In some embodiments,the GSIS responses resemble the GSIS response of endogenous human isletsto multiple glucose challenges. In some embodiments, the GSIS responseis observed immediately upon transplanting the cell into a human oranimal. In some embodiments, the GSIS response is observed withinapproximately 24 hours of transplanting the cell into a human or animal.In some embodiments, the GSIS response is observed within approximatelyone week of transplanting the cell into a human or animal. In someembodiments, the GSIS response is observed within approximately twoweeks of transplanting the cell into a human or animal. In someembodiments, the stimulation index of the cell as characterized by theratio of insulin secreted in response to high glucose concentrationscompared to low glucose concentrations is similar to the stimulationindex of an endogenous mature pancreatic β cell. In some embodiments,the SC-β cell exhibits a stimulation index of greater than 1. In someembodiments, the SC-β cell exhibits a stimulation index of greater thanor equal to 1. In some embodiments, the SC-β cell exhibits a stimulationindex of greater than 1.1. In some embodiments, the SC-β cell exhibits astimulation index of greater than or equal to 1.1. In some embodiments,the SC-β cell exhibits a stimulation index of greater than 2. In someembodiments, the SC-β cell exhibits a stimulation index of greater thanor equal to 2. In some embodiments, the SC-β cell exhibits a stimulationindex of at least 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5,4.6, 4.7, 4.8, 4.9, or 5.0 or greater.

In some embodiments, the SC-β cell exhibits cytokine-induced apoptosisin response to cytokines. In some embodiments, the SC-β cell exhibitscytokine-induced apoptosis in response to a cytokine selected from thegroup consisting of interleukin-1β (IL-β), interferon-γ (INF-γ), tumornecrosis factor-α (TNF-α), and combinations thereof.

In some embodiments, insulin secretion from the SC-β cell is enhanced inresponse to known anti-diabetic drugs (e.g., anti-diabetic drugs whichact on β cells ex vivo or in vitro, and/or anti-diabetic drugs generallyin vivo). The disclosure contemplates any known anti-diabetic drug. Insome embodiments, insulin secretion from the SC-β cell is enhanced inresponse to a secretagogue. In some embodiments, the secretagogue isselected from the group consisting of an incretin mimetic, asulfonylurea, a meglitinide, and combinations thereof.

In some embodiments, the SC-β cell is monohormonal. In some embodiments,the SC-β cell exhibits a morphology that resembles the morphology of anendogenous mature pancreatic β cell. In some embodiments, the SC-β cellencapsulates crystalline insulin granules. In some embodiments, the SC-βcell exhibits encapsulated crystalline insulin granules under electronmicroscopy that resemble insulin granules of an endogenous maturepancreatic β cell. In some embodiments, the SC-β cell exhibits a lowrate of replication. In some embodiments, the SC-β cell exhibits a lowrate of replication. In some embodiments, the SC-β cell exhibits a low,but increased rate of replication as measured by staining for C-peptideand Ki67 in response to treatment with prolactin.

In some embodiments, the SC-β cell increases intracellular Ca²⁺ inresponse to glucose. In some embodiments, the SC-β cell exhibits aglucose stimulated Ca²⁺ flux (GSCF) that resembles the GSCF of anendogenous mature pancreatic β cell. In some embodiments, the SC-β cellexhibits a GSCF response to at least three sequential glucose challengesin a manner that resembles the GSCF response of an endogenous maturepancreatic β cell to multiple glucose challenges.

In some embodiments, the SC-β cell expresses at least one markercharacteristic of an endogenous mature pancreatic β cell selected fromthe group consisting of insulin, C-peptide, PDX1, MAFA, NKX6-1, PAX6,NEUROD1, glucokinase (GCK), SLC2A1, PCSK1, KCNJ11, ABCC8, SLC30A8,SNAP25, RAB3A, GAD2, and PTPRN.

In some embodiments, the SC-β cell does not express at least one marker(e.g., a marker not expressed by endogenous mature pancreatic β cells)selected from the group consisting of a) a hormone selected from thegroup consisting of i) glucagon (GCG), and ii) somatostatin (SST); b) anacinar cell marker selected from the group consisting of i) amylase, andii) carboxypeptdase A (CPA1), c) an a cell marker selected from thegroup consisting of i) GCG, Arx, Irx1, and Irx2, d) a ductal cell markerselected from the group consisting of i) CFTR, and ii) Sox9.

The SC-β cells are differentiated in vitro from any starting cell as theinvention is not intended to be limited by the starting cell from whichthe SC-β cells are derived. Exemplary starting cells include, withoutlimitation, insulin-positive endocrine cells or any precursor thereofsuch as a Nkx6-1-positive pancreatic progenitor cell, a Pdx1-positivepancreatic progenitor cell, and a pluripotent stem cell, an embryonicstem cell, and induced pluripotent stem cell. In some embodiments, theSC-β cells are differentiated in vitro from a reprogrammed cell, apartially reprogrammed cell (i.e., a somatic cell, e.g., a fibroblastwhich has been partially reprogrammed such that it exists in anintermediate state between an induced pluripotency cell and the somaticcell from which it has been derived), a transdifferentiated cell. Insome embodiments, the SC-β cells disclosed herein can be differentiatedin vitro from an insulin-positive endocrine cell or a precursor thereof.In some embodiments, the SC-β cell is differentiated in vitro from aprecursor selected from the group consisting of a Nkx6-1-positivepancreatic progenitor cell, a Pdx1-positive pancreatic progenitor cell,and a pluripotent stem cell. In some embodiments, the pluripotent stemcell is selected from the group consisting of an embryonic stem cell andinduced pluripotent stem cell. In some embodiments, the SC-β cell or thepluripotent stem cell from which the SC-β cell is derived is human. Insome embodiments, the SC-β cell is human.

In some embodiments, the SC-β cell is not genetically modified. In someembodiments, the SC-β cell obtains the features it shares in common withnative β cells in the absence of a genetic modification of cells. Insome embodiments, the SC-β cell is genetically modified.

In some embodiments, the insulin produced per SC-β cell is at least 0.5μIU per 1000 cells per 30 minute incubation (e.g., ex vivo) at a highglucose concentration.

In some embodiments, the insulin produced per SC-β cell is at least 1,at least 2, at least 3, at least 4 at least 5 at least 6, at least 7 atleast 8 or at least 9 μIU per 1000 cells per 30 minute incubation at ahigh glucose concentration. In some embodiments, the insulin producedper SC-β cell is between 0.5 and 10 μIU per 1000 cells per 30 minuteincubation at a high glucose concentration. In some embodiments, theinsulin produced per SC-β cell is approximately 2.5 μIU per 1000 cellsper 30 minute incubation at a high glucose concentration.

In some aspects, the disclosure provides a cell line comprising a SC-βcell described herein. In some embodiments, the SC-β cells stablyexpress insulin. In some embodiments, the SC-β cell can be frozen,thawed, and amplified with a doubling time of 24 to 44 hours withoutsignificant morphological changes until at least 30 passages.

Generating SC-β Cells

Aspects of the disclosure relate to generating SC-β cells (e.g.,pancreatic β cells). Generally, the at least one SC-β cell or precursorthereof, e.g., pancreatic progenitors produced according to the methodsdisclosed herein can comprise a mixture or combination of differentcells, e.g., for example a mixture of cells such as a Pdx1-positivepancreatic progenitors, pancreatic progenitors co-expressing Pdx1 andNKX6-1, a Ngn3-positive endocrine progenitor cell, an insulin-positiveendocrine cell (e.g., a β-like cell), and an insulin-positive endocrinecell, and/or other pluripotent or stem cells.

The at least one SC-β cell or precursor thereof can be producedaccording to any suitable culturing protocol to differentiate a stemcell or pluripotent cell to a desired stage of differentiation. In someembodiments, the at least one SC-β cell or the precursor thereof areproduced by culturing at least one pluripotent cell for a period of timeand under conditions suitable for the at least one pluripotent cell todifferentiate into the at least one SC-β cell or the precursor thereof.

In some embodiments, the at least one SC-β cell or precursor thereof isa substantially pure population of SC-β cells or precursors thereof. Insome embodiments, a population of SC-β cells or precursors thereofcomprises a mixture of pluripotent cells or differentiated cells. Insome embodiments, a population SC-β cells or precursors thereof aresubstantially free or devoid of embryonic stem cells or pluripotentcells or iPS cells.

In some embodiments, a somatic cell, e.g., fibroblast can be isolatedfrom a subject, for example as a tissue biopsy, such as, for example, askin biopsy, and reprogrammed into an induced pluripotent stem cell forfurther differentiation to produce the at least one SC-β cell orprecursor thereof for use in the compositions and methods describedherein. In some embodiments, a somatic cell, e.g., fibroblast ismaintained in culture by methods known by one of ordinary skill in theart, and in some embodiments, propagated prior to being converted intoSC-β cells by the methods as disclosed herein.

In some embodiments, the at least one SC-β cell or precursor thereof aremaintained in culture by methods known by one of ordinary skill in theart, and in some embodiments, propagated prior to being converted intoSC-β cells by the methods as disclosed herein.

Further, at least one SC-β cell or precursor thereof, e.g., pancreaticprogenitor can be from any mammalian species, with non-limiting examplesincluding a murine, bovine, simian, porcine, equine, ovine, or humancell. For clarity and simplicity, the description of the methods hereinrefers to a mammalian at least one SC-β cell or precursor thereof but itshould be understood that all of the methods described herein can bereadily applied to other cell types of at least one SC-β cell orprecursor thereof. In some embodiments, the at least one SC-β cell orprecursor thereof is derived from a human individual.

Inducing the Differentiation of Pluripotent Stem Cells to DefinitiveEndoderm Cells

Aspects of the disclosure involve definitive endoderm cells. Definitiveendoderm cells of use herein can be derived from any source or generatedin accordance with any suitable protocol. In some aspects, pluripotentstem cells, e.g., iPSCs or hESCs, are differentiated to endoderm cells.In some aspects, the endoderm cells are further differentiated, e.g., toprimitive gut tube cells, Pdx1-positive pancreatic progenitor cells,NKX6-1-positive pancreatic progenitor cells, Ngn3-positive endocrineprogenitor cells, or insulin-positive endocrine cells, followed byinduction or maturation to SC-β cells.

In some embodiments, the stem cells may be plated onto a new substrateor the medium may be exchanged to remove extracellular matrix or solublefactors that inhibit differentiation. This is sometimes referred to asthe “direct differentiation method”, and is described in general termsin International patent publication WO 01/51616, and U.S. PatentPublication 2002/0019046, which is incorporated herein in its entiretyby reference. It is usually preferable in the direct differentiationmethod to begin with a feeder-free culture of stem cells, so as to avoidpotential complications in the differentiation process caused byresidual feeder cells. Another approach is to put undifferentiated stemcells in suspension culture, which will frequently cause them to formaggregates of differentiated and undifferentiated cells. For example,stem cells can be harvested by brief collagenase digestion, dissociatedinto clusters, and passaged in non-adherent cell culture plates. Theaggregates can be fed every few days, and then harvested after asuitable period, typically 4-8 days. Depending on the conditions,aggregates generally start by forming a heterogeneous population of celltypes, including a substantial frequency of endoderm cells. Theaggregates can then be dispersed and replated for the next stage in thedifferentiation process, on substrates such as laminin or fibronectin;or passaged in suspension culture using, for example, non-adherentplates and a suitable medium.

Direct differentiation or differentiation in aggregates can be monitoredfor the presence of endoderm cells using suitable markers such as thoselisted in U.S. Pat. No. 7,326,572. In some preferred embodiments,differentiation can be monitored for the presence of endoderm cellsusing markers such as Sox17. Once a sufficient proportion of endoderm isobtained, cells can be replated or otherwise manipulated to beginanother stage of differentiation. In certain circumstances,differentiation or maintenance of cells may be enhanced if the cells arekept in micromass clusters (for example, 50 to 5,000 cells). Additionalstages of differentiation contemplated by the disclosure are shown inFIG. 1.

In some embodiments, definitive endoderm cells are produced bycontacting (e.g., culturing) a pluripotent stem cell with a compound ofFormula (I) as described in U.S. Pat. No. 8,507,274 (“the '274 patent”),incorporated by reference herein. Compound with Formula (I) as describedin the '274 patent are cell permeable small molecules, and can controlcellular processes by modulating signal transduction pathways, geneexpression or metabolism and have been effectively used in stem celldifferentiation protocols. Small molecules can be synthesized in highquantity and purity as well as conveniently supplied or removed, givingthem great potential to be useful for therapeutic applications. Highthroughput screens have been performed to identify novel small moleculesthat can support the self renewal of ES cells (Chen et al., 2006;Desbordes et al., 2008), cardiogenic specification of mouse ES cells (Wuet al., 2004) or neural progenitor cells (Diamandis et al., 2007) aswell as inducing specific cell types, notably neuronal and muscle cells(reviewed by (Ding and Schultz, 2004). It is expected that compounds ofFormula (I) from the '274 patent can be used to differentiate apluripotent stem cell to a definitive endoderm cell.

In some embodiments, the compound of Formula (I) from the '274 patentcomprises:

wherein:

R¹ and R² are independently H, alkyl, alkenyl, alkynyl, aryl,heteroaryl, cyclyl, or cyclyl, each of which can be optionallysubstituted and/or can be interrupted in the backbone with one or moreof O, N, S, S(O), and C(O);

R³ and R⁴ are independently H, halogen, alkyl, alkenyl, alkynyl, alkoxy,aryl, heteroaryl, cyclyl, or cyclyl, each of which can be optionallysubstituted, or R³ and R⁴ together with the carbon to which they areattached from an optionally substituted cyclyl of heterocycyl; and

Lis C₁-C₁₀ alkylenyl, C₂-C₁₀ alkenylenyl, or C₂-C₁₀ alkynylenyl, each ofwhich can be optionally substituted and/or can be interrupted in thebackbone with one or more of O, N, S, S(O), and C(O).

In some embodiments, the compound of Formula (I) from the '274 patentcomprises IDE1 below:

In some embodiments, the compound of Formula (I) from the '274 patentcomprises IDE2 below:

The '274 patent describes methods for confirming the identity of thedefinitive endoderm cell thus derived, as well as methods for isolating,storing, expanding, and further differentiating definitive endoderm,which can all be used with the compositions and methods describedherein, as will be appreciated by the skilled artisan.

In some embodiments, definitive endoderm cells can be obtained bydifferentiating at least some pluripotent cells in a population intodefinitive endoderm cells, e.g., by contacting a population ofpluripotent cells with i) at least one growth factor from the TGF-βsuperfamily, and ii) a WNT signaling pathway activator, to induce thedifferentiation of at least some of the pluripotent cells intodefinitive endoderm cells, wherein the definitive endoderm cells expressat least one marker characteristic of definitive endoderm.

The disclosure contemplates the use of any growth factor from the TGF-βsuperfamily that induces the pluripotent stem cells to differentiateinto definitive endoderm cells (e.g., alone, or in combination with aWNT signaling pathway activator). In some embodiments, the at least onegrowth factor from the TGF-β superfamily comprises Activin A. In someembodiments, the at least one growth factor from the TGF-β superfamilycomprises growth differentiating factor 8 (GDF8).

The disclosure contemplates the use of any WNT signaling pathwayactivator that induces the pluripotent stem cells to differentiate intodefinitive endoderm cells (e.g., alone, or in combination with a growthfactor from the TGF-β superfamily). In some embodiments, the WNTsignaling pathway activator comprises CHIR99021. In some embodiments,the WNT signaling pathway activator comprises Wnt3a recombinant protein.

The skilled artisan will appreciate that the concentrations of agents(e.g., growth factors) employed may vary. In some embodiments, thepluripotent cells are contacted with the at least one growth factor fromthe TGF-β superfamily at a concentration of between 10 ng/mL-1000 ng/mL.In some embodiments, the pluripotent cells are contacted with the atleast one growth factor from the TGF-β superfamily at a concentration of100 ng/mL. In some embodiments, the pluripotent cells are contacted withthe at least one growth factor from the TGF-β superfamily at aconcentration of 20 ng/mL, 30 ng/mL, 40 ng/mL, 50 ng/mL, 60 ng/mL, 70ng/mL, 80 ng/mL, or 90 ng/mL. In some embodiments, the pluripotent cellsare contacted with the at least one growth factor from the TGF-βsuperfamily at a concentration of 91 ng/mL, 92 ng/mL, 93 ng/mL, 94ng/mL, 95 ng/mL, 96 ng/mL, 97 ng/mL, 98 ng/mL or 99 ng/mL. In someembodiments, the pluripotent cells are contacted with the at least onegrowth factor from the TGF-β superfamily at a concentration of 110ng/mL, 120 ng/mL, 130 ng/mL, 140 ng/mL, 150 ng/mL, 160 ng/mL, 170 ng/mL,180 ng/mL, or 190 ng/mL. In some embodiments, the pluripotent cells arecontacted with the at least one growth factor from the TGF-β superfamilyat a concentration of 101 ng/mL, 102 ng/mL, 103 ng/mL, 104 ng/mL, 105ng/mL, 106 ng/mL, 107 ng/mL, 108 ng/mL or 109 ng/mL.

In some embodiments, the pluripotent cells are contacted with the WNTsignaling pathway activator at a concentration of between 1.4 μg/mL-140μg/mL. In some embodiments, the pluripotent cells are contacted with theWNT signaling pathway activator at a concentration of 14 μg/mL. In someembodiments, the pluripotent cells are contacted with the WNT signalingpathway activator at a concentration of 2 μg/mL, 3 μg/mL, 4 μg/mL, 5μg/mL, 6 μg/mL, 7 μg/mL, 8 μg/mL, 9 μg/mL, 10 μg/mL, 11 μg/mL, 12 μg/mLor 13 μg/mL. In some embodiments, the pluripotent cells are contactedwith the WNT signaling pathway activator at a concentration of 15 μg/mL,16 μg/mL, 17 μg/mL, 18 μg/mL, 19 μg/mL, 20 μg/mL, 21 μg/mL, 22 μg/mL, 23μg/mL, 24 μg/mL, 25 μg/mL, 26 μg/mL, 27 μg/mL, 28 μg/mL, 29 μg/mL, or 30μg/mL. In some embodiments, the pluripotent cells are contacted with theWNT signaling pathway activator at a concentration of 13.1 μg/mL, 13.2μg/mL, 13.3 μg/mL, 13.4 μg/mL, 13.5 μg/mL, 13.6 μg/mL, 13.7 μg/mL, 13.8μg/mL, or 13.9 μg/mL. In some embodiments, the pluripotent cells arecontacted with the WNT signaling pathway activator at a concentration of14.1 μg/mL, 14.2 μg/mL, 14.3 μg/mL, 14.4 μg/mL, 14.5 μg/mL, 14.6 μg/mL,14.7 μg/mL, 14.8 μg/mL, or 14.9 μg/mL.

Generally, the pluripotent cells are maintained in suitable culturemedium (e.g., suspension culture) for a period of time sufficient toinduce the differentiation of at least some of the pluripotent cellsinto definitive endoderm cells. An exemplary suitable culture medium isshown in Table 1 below.

TABLE 1 Agent Amount MCDB131 1 L Glucose 0.44 NaHCO3 2.46 g FAF-BSA 20 gITS-X 20 uL GlutaMAX ™ 10 mL Vitamin C 0.044 g Heparin 0 g P/S 10 mL

In some embodiments, a suitable culture medium for differentiatingpluripotent cells into definitive endoderm cells comprises S1 media.

In some embodiments, contacting the pluripotent cells is effected insuspension culture. In some embodiments, the suspension culture ismaintained in a spinner flask. In some embodiments, the period of timeis 3 days. In some embodiments, the at least one growth factor from theTGF-β superfamily, and WNT signaling pathway activator are added to thesuspension culture on the first day. In some embodiments, the at leastone growth factor from the TGF-β superfamily is replenished in thesuspension culture on the second day. In some embodiments, the WNTsignaling pathway activator is not replenished in the suspension cultureon the second day. In some embodiments, the WNT signaling pathwayactivator is removed from the suspension culture on the second day. Insome embodiments, the at least one growth factor from the TGF-βsuperfamily is replenished in the suspension culture on the second day,and the WNT signaling pathway activator is removed from the suspensionculture or not replenished in the suspension culture on the second day.In some embodiments, neither the at least one growth factor from theTGF-β superfamily or the WNT signaling pathway activator are replenishedin the suspension culture on the third day. In some embodiments, boththe at least one growth factor from the TGF-β superfamily and the WNTsignaling pathway activator are removed from the suspension culture onthe third day.

The methods are capable of inducing the differentiation of at least onepluripotent cell in a population of cells into a definitive endodermcell. Generally, any pluripotent cell can be differentiated into adefinitive endoderm cell using a method described herein. In someembodiments, the pluripotent cells comprise induced pluripotent stemcells. In some embodiments, the pluripotent cells comprise embryonicstem cells. In some embodiments, the pluripotent cells comprise humancells.

In some embodiments, differentiating at least some pluripotent cells ina population into definitive endoderm cells is achieved by a process ofcontacting a population of pluripotent cells with i) Activin A, and ii)CHIR99021, to induce the differentiation of at least some of thepluripotent cells in the population into definitive endoderm cells,wherein the definitive endoderm cells express at least one markercharacteristic of definitive endoderm.

Other methods for producing definitive endoderm cells are known in theart, including, for example the methods which are set forth in UnitedStates application publication US2006/0003446 to G. Keller, et al.;US2006/0003313 to K. D'Amour, et al., US2005/0158853 to K. D'Amour, etal., and US2005/0260749 of Jon Odorico, et al., relevant portions ofwhich are incorporated by reference herein.

In some embodiments, a definitive endoderm cell produced by the methodsas disclosed herein expresses at least one marker selected from thegroup consisting of: Nodal, Tmprss2, Tmem30b, St14, Spink3, Sh3gl2,Ripk4, Rab15, Npnt, Clic6, Cldn8, Cacna1b, Bnip1, Anxa4, Emb, FoxA1,Sox17, and Rbm35a, wherein the expression of at least one marker isupregulated to by a statistically significant amount in the definitiveendoderm cell relative to the pluripotent stem cell from which it wasderived. In some embodiments, a definitive endoderm cell produced by themethods as disclosed herein does not express by a statisticallysignificant amount at least one marker selected the group consisting of:Gata4, SPARC, AFP and Dab2 relative to the pluripotent stem cell fromwhich it was derived. In some embodiments, a definitive endoderm cellproduced by the methods as disclosed herein does not express by astatistically significant amount at least one marker selected the groupconsisting of: Zic1, Pax6, Flk1 and CD31 relative to the pluripotentstem cell from which it was derived.

In some embodiments, a definitive endoderm cell produced by the methodsas disclosed herein has a higher level of phosphorylation of Smad2 by astatistically significant amount relative to the pluripotent stem cellfrom which it was derived. In some embodiments, a definitive endodermcell produced by the methods as disclosed herein has the capacity toform gut tube in vivo. In some embodiments, a definitive endoderm cellproduced by the methods as disclosed herein can differentiate into acell with morphology characteristic of a gut cell, and wherein a cellwith morphology characteristic of a gut cell expresses FoxA2 and/orClaudin6. In some embodiments, a definitive endoderm cell produced bythe methods as disclosed herein can be further differentiated into acell of endoderm origin.

In some embodiments, a population of pluripotent stem cells are culturedin the presence of at least one β cell maturation factor prior to anydifferentiation or during the first stage of differentiation. One canuse any pluripotent stem cell, such as a human pluripotent stem cell, ora human iPS cell or any of pluripotent stem cell as discussed herein orother suitable pluripotent stem cells. In some embodiments, a β cellmaturation factor as described herein can be present in the culturemedium of a population of pluripotent stem cells or may be added inbolus or periodically during growth (e.g. replication or propagation) ofthe population of pluripotent stem cells. In certain examples, apopulation of pluripotent stem cells can be exposed to at least one βcell maturation factor prior to any differentiation. In other examples,a population of pluripotent stem cells may be exposed to at least one βcell maturation factor during the first stage of differentiation.

Inducing the Differentiation of Definitive Endoderm Cells to PrimitiveGut Tube Cells

Aspects of the disclosure involve primitive gut tube cells. Primitivegut tube cells of use herein can be derived from any source or generatedin accordance with any suitable protocol. In some aspects, definitiveendoderm cells are differentiated to primitive gut tube cells. In someaspects, the primitive gut tube cells are further differentiated, e.g.,to Pdx1-positive pancreatic progenitor cells, NKX6-1-positive pancreaticprogenitor cells, Ngn3-positive endocrine progenitor cells,insulin-positive endocrine cells, followed by induction or maturation toSC-β cells.

In some embodiments, primitive gut tube cells can be obtained bydifferentiating at least some definitive endoderm cells in a populationinto primitive gut tube cells, e.g., by contacting definitive endodermcells with at least one growth factor from the fibroblast growth factor(FGF) family, to induce the differentiation of at least some of thedefinitive endoderm cells into primitive gut tube cells, wherein theprimitive gut tube cells express at least one marker characteristic ofprimitive gut tube cells.

The disclosure contemplates the use of any growth factor from the FGFfamily that induces definitive endoderm cells to differentiate intoprimitive gut tube cells (e.g., alone, or in combination with otherfactors). In some embodiments, the at least one growth factor from theFGF family comprises keratinocyte growth factor (KGF). In someembodiments, the at least one growth factor from the FGF familycomprises FGF2. In some embodiments, the at least one growth factor fromthe FGF family comprises FGF8B. In some embodiments, the at least onegrowth factor from the FGF family comprises FGF10. In some embodiments,the at least one growth factor from the FGF family comprises FGF21.

The skilled artisan will appreciate that the concentrations of growthfactor employed may vary. In some embodiments, the definitive endodermcells are contacted with the at least one growth factor from the FGFfamily at a concentration of between 5 ng/mL-500 ng/mL. In someembodiments, the definitive endoderm cells are contacted with the atleast one growth factor from the FGF family at a concentration of 10ng/mL, 15 ng/mL, 20 ng/mL, 25 ng/mL, 30 ng/mL, 35 ng/mL, or 40 ng/mL. Insome embodiments, the definitive endoderm cells are contacted with theat least one growth factor from the FGF family at a concentration of 60ng/mL, 65 ng/mL, 70 ng/mL, 75 ng/mL, 80 ng/mL, 85 ng/mL, 90 ng/mL, 95ng/mL or 100 ng/mL. In some embodiments, the definitive endoderm cellsare contacted with the at least one growth factor from the FGF family ata concentration of 41 ng/mL, 42 ng/mL, 43 ng/mL, 44 ng/mL, 45 ng/mL, 46ng/mL, 47 ng/mL, 48 ng/mL or 49 ng/mL. In some embodiments, thedefinitive endoderm cells are contacted with the at least one growthfactor from the FGF family at a concentration of 51 ng/mL, 52 ng/mL, 53ng/mL, 54 ng/mL, 55 ng/mL, 56 ng/mL, 57 ng/mL, 58 ng/mL or 59 ng/mL. Insome embodiments, the definitive endoderm cells are contacted with theat least one growth factor from the FGF family at a concentration of 50ng/mL.

In some embodiments, the definitive endoderm cells are cultured in asuitable culture medium.

Generally, the definitive endoderm cells are maintained in a suitableculture medium (e.g., suspension culture) for a period of timesufficient to induce the differentiation of at least some of thedefinitive endoderm cells into primitive gut tube cells. An exemplarysuitable culture medium is shown in Table 2 below.

TABLE 2 Agent Amount MCDB131 1 L Glucose 0.44 g NaHCO3 1.23 g FAF-BSA 20g ITS-X 20 uL GlutaMAX ™ 10 mL Vitamin C 0.044 g Heparin 0 g P/S 10 mL

In some embodiments, a suitable culture medium for differentiatingdefinitive endoderm cells into primitive gut tube cells comprises S2media.

In some embodiments, contacting the definitive endoderm cells iseffected in suspension culture. In some embodiments, the suspensionculture is maintained in a spinner flask. In some embodiments, theperiod of time is between 2 days and 5 days. In some embodiments, theperiod of time is 3 days. In some embodiments, the suspension culture isreplenished every other day.

In some embodiments, definitive endoderm cells can be obtained bydifferentiating at least some of the definitive endoderm cells in apopulation into primitive gut tube cells, e.g., by contacting thedefinitive endoderm cells with KGF, to induce the differentiation of atleast some of the definitive endoderm cells into primitive gut tubecells, wherein the primitive gut tube cells express at least one markercharacteristic of definitive endoderm.

Inducing the Differentiation of Primitive Gut Tube Cells toPdx1-Positive Pancreatic Progenitor Cells

Aspects of the disclosure involve Pdx1-positive pancreatic progenitorcells. Pdx1-positive pancreatic progenitor cells of use herein can bederived from any source or generated in accordance with any suitableprotocol. In some aspects, primitive gut tube cells are differentiatedto Pdx1-positive pancreatic progenitor cells. In some aspects, thePdx1-positive pancreatic progenitor cells are further differentiated,e.g., NKX6-1-positive pancreatic progenitor cells, Ngn3-positiveendocrine progenitor cells, insulin-positive endocrine cells, followedby induction or maturation to SC-β cells.

In some aspects, Pdx1-positive pancreatic progenitor cells can beobtained by differentiating at least some primitive gut tube cells in apopulation into Pdx1-positive pancreatic progenitor cells, e.g., bycontacting primitive gut tube cells with i) at least one bonemorphogenic protein (BMP) signaling pathway inhibitor, ii) at least onegrowth factor from the FGF family, iii) at least one SHH pathwayinhibitor, iv) at least one retinoic acid (RA) signaling pathwayactivator; and v) at least one protein kinase C activator, to induce thedifferentiation of at least some of the primitive gut tube cells intoPdx1-positive pancreatic progenitor cells, wherein the Pdx1-positivepancreatic progenitor cells express Pdx1.

The disclosure contemplates the use of any BMP signaling pathwayinhibitor that induces primitive gut tube cells to differentiate intoPdx1-positive pancreatic progenitor cells (e.g., alone, or with anycombination of at least one growth factor from the FGF family, at leastone SHH pathway inhibitor, at least one retinoic acid signaling pathwayactivator, and at least one protein kinase C activator). In someembodiments, the BMP signaling pathway inhibitor comprises LDN193189.

The disclosure contemplates the use of any growth factor from the FGFfamily that induces primitive gut tube cells to differentiate intoPdx1-positive pancreatic progenitor cells (e.g., alone, or with anycombination of at least one BMP signaling pathway inhibitor, at leastone SHH pathway inhibitor, at least one retinoic acid signaling pathwayactivator, and at least one protein kinase C activator). In someembodiments, the at least one growth factor from the FGF familycomprises keratinocyte growth factor (KGF). In some embodiments, the atleast one growth factor from the FGF family is selected from the groupconsisting of FGF2, FGF8B, FGF10, and FGF21.

The disclosure contemplates the use of any SHH pathway inhibitor thatinduces primitive gut tube cells to differentiate into Pdx1-positivepancreatic progenitor cells (e.g., alone, or with any combination of atleast one BMP signaling pathway inhibitor, at least one growth factorfrom the FGF family, at least one retinoic acid signaling pathwayactivator, and at least one protein kinase C activator). In someembodiments, the SHH pathway inhibitor comprises Sant1.

The disclosure contemplates the use of any RA signaling pathwayactivator that induces primitive gut tube cells to differentiate intoPdx1-positive pancreatic progenitor cells (e.g., alone, or with anycombination of at least one BMP signaling pathway inhibitor, at leastone growth factor from the FGF family, at least one SHH pathwayinhibitor, and at least one protein kinase C activator). In someembodiments, the RA signaling pathway activator comprises retinoic acid.

The disclosure contemplates the use of any PKC activator that inducesprimitive gut tube cells to differentiate into Pdx1-positive pancreaticprogenitor cells (e.g., alone, or with any combination of at least oneBMP signaling pathway inhibitor, at least one growth factor from the FGFfamily, at least one SHH pathway inhibitor, and at least one RAsignaling pathway activator). In some embodiments, the PKC activatorcomprises PdbU. In some embodiments, the PKC activator comprises TPB.

The skilled artisan will appreciate that the concentrations of agents(e.g., growth factors) employed may vary. In some embodiments, theprimitive gut tube cells are contacted with the BMP signaling pathwayinhibitor at a concentration of between 20 nM-2000 nM. In someembodiments, the primitive gut tube cells are contacted with the BMPsignaling pathway inhibitor at a concentration of 30 40 nM, 50 nM, 60nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM,160 nM, 170 nM, 180 nM, or 190 nM. In some embodiments, the primitivegut tube cells are contacted with the BMP signaling pathway inhibitor ata concentration of 191 nM, 192 nM, 193 nM, 194 nM, 195 nM, 196 nM, 197nM, 198 nM, or 199 nM. In some embodiments, the primitive gut tube cellsare contacted with the BMP signaling pathway inhibitor at aconcentration of 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM,1000 nM, 1100 nM, 1200 nM, 1300 nM, 1400 nM, 1500 nM, 1600 nM, 1700 nM,1800 nM, or 1900 nM. In some embodiments, the primitive gut tube cellsare contacted with the BMP signaling pathway inhibitor at aconcentration of 210 nM, 220 nM, 230 nM, 240 nM, 250 nM, 260 nM, 270 nM,280 nM, or 290 nM. In some embodiments, the primitive gut tube cells arecontacted with the BMP signaling pathway inhibitor at a concentration of200 nM.

In some embodiments, the primitive gut tube cells are contacted with theat least one growth factor from the FGF family at a concentration ofbetween 5 ng/mL-500 ng/mL. In some embodiments, the primitive gut tubecells are contacted with the at least one growth factor from the FGFfamily at a concentration of 10 ng/mL, 15 ng/mL, 20 ng/mL, 25 ng/mL, 30ng/mL, 35 ng/mL, or 40 ng/mL. In some embodiments, the primitive guttube cells are contacted with the at least one growth factor from theFGF family at a concentration of 60 ng/mL, 65 ng/mL, 70 ng/mL, 75 ng/mL,80 ng/mL, 85 ng/mL, 90 ng/mL, 95 ng/mL or 100 ng/mL. In someembodiments, the primitive gut tube cells are contacted with the atleast one growth factor from the FGF family at a concentration of 41ng/mL, 42 ng/mL, 43 ng/mL, 44 ng/mL, 45 ng/mL, 46 ng/mL, 47 ng/mL, 48ng/mL or 49 ng/mL. In some embodiments, the primitive gut tube cells arecontacted with the at least one growth factor from the FGF family at aconcentration of 51 ng/mL, 52 ng/mL, 53 ng/mL, 54 ng/mL, 55 ng/mL, 56ng/mL, 57 ng/mL, 58 ng/mL or 59 ng/mL. In some embodiments, theprimitive gut tube cells are contacted with the at least one growthfactor from the FGF family at a concentration of 50 ng/mL.

In some embodiments, the primitive gut tube cells are contacted with theat least one SHH pathway inhibitor at a concentration of between 0.1 μMand 0.5 μM. In some embodiments, the primitive gut tube cells arecontacted with the at least one SHH pathway inhibitor at a concentrationof 0.11 μM, 0.12 μM, 0.13 μM, 0.14 μM, 0.15 μM, 0.16 μM, 0.17 μM, 0.18μM, 0.19 μM, 0.2 μM, 0.21 μM, 0.22 μM, 0.23 μM, or 0.24 μM. In someembodiments, the primitive gut tube cells are contacted with the atleast one SHH pathway inhibitor at a concentration of 0.26 μM, 0.27 μM,0.28 μM, 0.29 μM, 0.30 μM, 0.31 μM, 0.32 μM, 0.33 μM, 0.34 μM, 0.35 μM,0.36 μM, 0.37 μM, 0.38 μM, 0.39 μM, 0.40 μM, 0.41 μM, 0.42 μM, 0.43 μM,0.44 μM, 0.45 μM, 0.46 μM, 0.47 μM, 0.48 μM, 0.49 μM. In someembodiments, the primitive gut tube cells are contacted with the atleast one SHH pathway inhibitor at a concentration of 0.25 μM.

In some embodiments, the primitive gut tube cells are contacted with theRA signaling pathway activator at a concentration of between 0.01 μM-1.0μM. In some embodiments, the primitive gut tube cells are contacted withthe RA signaling pathway activator at a concentration of 0.02 μM, 0.03μM, 0.04 μM, 0.05 μM, 0.06 μM, 0.07 μM, 0.08 μM, or 0.09 μM. In someembodiments, the primitive gut tube cells are contacted with the RAsignaling pathway activator at a concentration of 0.20 μM, 0.30 μM, 0.40μM, 0.05 μM, 0.60 μM, 0.70 μM, 0.80 μM, or 0.90 μM. In some embodiments,the primitive gut tube cells are contacted with the RA signaling pathwayactivator at a concentration of 0.1 μM.

In some embodiments, the primitive gut tube cells are contacted with PKCactivator at a concentration of between 50 nM-5000 nM. In someembodiments, the primitive gut tube cells are contacted with the PKCactivator at a concentration of 100 nM, 150 nM, 200 nM, 250 nM, 300 nM,350 nM, 400 nM, 450 nM, 460 nM, 470 nM, 480 nM, or 490 nM. In someembodiments, the primitive gut tube cells are contacted with the PKCactivator at a concentration of 491 nM, 492 nM, 493 nM, 494 nM, 495 nM,496 nM, 497 nM, 498 nM, or 499 nM. In some embodiments, the primitivegut tube cells are contacted with the PKC activator at a concentrationof 600 nM, 700 nM, 800 nM, 900 nM, 1000 nM, 1100 nM, 1200 nM, 1300 nM,1400 nM, 1500 nM, 1600 nM, 1700 nM, 1800 nM, 1900 nM, or 2000 nM. Insome embodiments, the primitive gut tube cells are contacted with thePKC activator at a concentration of 501 nM, 502 nM, 503 nM, 504 nM, 505nM, 506 nM, 507 nM, 508 nM, or 509 nM, 510 nM, 520 nM, 530 nM, 540 nM,550 nM, 560 nM, 570 nM, 580 nM, or 590 nM. In some embodiments, theprimitive gut tube cells are contacted with the PKC activator at aconcentration of 500 nM.

Generally, the primitive gut tube cells are maintained in a suitableculture medium (e.g., suspension culture) for a period of timesufficient to induce the differentiation of at least some of theprimitive gut tube cells into Pdx1-positive pancreatic progenitor cells.An exemplary suitable culture medium is shown in Table 3 below.

TABLE 3 Agent Amount MCDB131 1 L Glucose 0.44 g NaHCO3 1.23 FAF-BSA 20 gITS-X 5 mL GlutaMAX ™ 10 mL Vitamin C 0.044 g Heparin 0 g P/S 10 mL

In some embodiments, S3 media can be used as a suitable culture mediumfor differentiating primitive gut tube cells into pancreatic progenitorcells.

In some embodiments, contacting the primitive gut tube cells is effectedin suspension culture. In some embodiments, the suspension culture ismaintained in a spinner flask. In some embodiments, the period of timeis at least 2 days. In some embodiments, the suspension culture isreplenished every day.

In some embodiments, primitive gut tube cells can be obtained bydifferentiating at least some of the primitive gut tube cells in apopulation into Pdx1-positive pancreatic progenitor cells, e.g., bycontacting the primitive gut tube cells with i) LDN193189, ii) KGF, iii)Sant1; iv) RA; and iv) PdbU, to induce the differentiation of at leastsome of the primitive gut tube cells into Pdx1-positive pancreaticprogenitor cells, wherein the Pdx1-positive pancreatic progenitor cellsexpress Pdx1.

Inducing the Differentiation of Pdx1-Positive Pancreatic ProgenitorCells to NKX6-1+ Pancreatic Progenitor Cells

Aspects of the disclosure involve NKX6-1-positive pancreatic progenitorcells. NKX6-1-positive pancreatic progenitor cells of use herein can bederived from any source or generated in accordance with any suitableprotocol. In some aspects, Pdx1-positive pancreatic progenitor cells aredifferentiated to NKX6-1-positive pancreatic progenitor cells. In someaspects, the NKX6-1-positive pancreatic progenitor cells are furtherdifferentiated, e.g., to Ngn3-positive endocrine progenitor cells, orinsulin-positive endocrine cells, followed by induction or maturation toSC-β cells.

In some aspects, a method of producing a NKX6-1-positive pancreaticprogenitor cell from a Pdx1-positive pancreatic progenitor cellcomprises contacting a population of cells (e.g., under conditions thatpromote cell clustering) comprising Pdx1-positive pancreatic progenitorcells with at least two β cell-maturation factors comprising a) at leastone growth factor from the fibroblast growth factor (FGF) family, b) asonic hedgehog pathway inhibitor, and optionally c) a low concentrationof a retinoic acid (RA) signaling pathway activator, for a period of atleast five days to induce the differentiation of at least onePdx1-positive pancreatic progenitor cell in the population intoNKX6-1-positive pancreatic progenitor cells, wherein the NKX6-1-positivepancreatic progenitor cells expresses NKX6-1.

In some embodiments, the Pdx1-positive, NKX6-1-positive pancreaticprogenitor cells are obtained by contacting Pdx1-positive pancreaticprogenitor cells under conditions that promote cell clustering with i)at least one growth factor from the FGF family, ii) at least one SHHpathway inhibitor, and optionally iii) low concentrations of a RAsignaling pathway activator, for a period of five days to induce thedifferentiation of at least some of the Pdx1-positive pancreaticprogenitor cells into Pdx1-positive, NKX6-1-positive pancreaticprogenitor cells, wherein the Pdx1-positive, NKX6-1-positive pancreaticprogenitor cells expresses Pdx1 and NKX6-1.

In some embodiments, the Pdx1-positive, NKX6-1-positive pancreaticprogenitor cells are obtained by contacting Pdx1-positive pancreaticprogenitor cells with i) at least one growth factor from the FGF family,ii) at least one SHH pathway inhibitor, and iii) a RA signaling pathwayactivator, to induce the differentiation of at least some of thePdx1-positive pancreatic progenitor cells into Pdx1-positive,NKX6-1-positive pancreatic progenitor cells, wherein the Pdx1-positive,NKX6-1-positive pancreatic progenitor cells expresses Pdx1 and NKX6-1.

In some embodiments, the Pdx1-positive pancreatic progenitor cells areproduced from a population of pluripotent cells. In some embodiments,the Pdx1-positive pancreatic progenitor cells are produced from apopulation of iPS cells. In some embodiments, the Pdx1-positivepancreatic progenitor cells are produced from a population of ESC cells.In some embodiments, the Pdx1-positive pancreatic progenitor cells areproduced from a population of definitive endoderm cells. In someembodiments, the Pdx1-positive pancreatic progenitor cells are producedfrom a population of primitive gut tube cells.

The disclosure contemplates the use of any growth factor from the FGFfamily that induces Pdx1-positive pancreatic progenitor cells todifferentiate into NKX6-1-positive pancreatic progenitor cells (e.g.,alone, or with any combination of at least one SHH pathway inhibitor, oroptionally at least one retinoic acid signaling pathway activator). Insome embodiments, the at least one growth factor from the FGF familycomprises keratinocyte growth factor (KGF). In some embodiments, the atleast one growth factor from the FGF family is selected from the groupconsisting of FGF2, FGF8B, FGF10, and FGF21.

The disclosure contemplates the use of any SHH pathway inhibitor thatinduces Pdx1-positive pancreatic progenitor cells to differentiate intoNKX6-1-positive pancreatic progenitor cells (e.g., alone, or with anycombination of at least one growth factor from the FGF family, or atleast one retinoic acid signaling pathway activator). In someembodiments, the SHH pathway inhibitor comprises Sant1.

The disclosure contemplates the use of any RA signaling pathwayactivator that induces Pdx1-positive pancreatic progenitor cells todifferentiate into NKX6-1-positive pancreatic progenitor cells (e.g.,alone, or with any combination of at least one growth factor from theFGF family, and at least one SHH pathway inhibitor). In someembodiments, the RA signaling pathway activator comprises retinoic acid.

In some embodiments, the method comprises contacting the population ofcells (e.g., Pdx1-positive pancreatic progenitor cells) with at leastone additional β cell-maturation factor. In some embodiments, the atleast one additional β cell-maturation factor comprises at least onegrowth factor from the EGF family. In some embodiments, the methodcomprises contacting the Pdx1-positive pancreatic progenitor cells withat least one growth factor from the EGF family. The disclosurecontemplates the use of any growth factor from the EGF family thatfacilitates the differentiation of Pdx1-positive pancreatic progenitorcells into NKX6-1-positive pancreatic progenitor cells (e.g., togetherwith any combination of at least one growth factor from the FGF family,at least one SHH pathway inhibitor, and optionally at least one RAsignaling pathway activator). In some embodiments, the at least onegrowth factor from the EGF family comprises betacellulin. In someembodiments, the at least one growth factor from the EGF familycomprises EGF.

The skilled artisan will appreciate that the concentrations of agents(e.g., growth factors) employed may vary. In some embodiments, thePdx1-positive pancreatic progenitor cells are contacted with the atleast one growth factor from the FGF family at a concentration ofbetween 1 ng/mL-100 ng/mL. In some embodiments, the Pdx1-positivepancreatic progenitor cells are contacted with the at least one growthfactor from the FGF family at a concentration of 5 ng/mL, 10 ng/mL, 15ng/mL, 20 ng/mL, 25 ng/mL, 30 ng/mL, 35 ng/mL, or 40 ng/mL. In someembodiments, the Pdx1-positive pancreatic progenitor cells are contactedwith the at least one growth factor from the FGF family at aconcentration of 60 ng/mL, 65 ng/mL, 70 ng/mL, 75 ng/mL, 80 ng/mL, 85ng/mL, 90 ng/mL, 95 ng/mL or 100 ng/mL. In some embodiments, thePdx1-positive pancreatic progenitor cells are contacted with the atleast one growth factor from the FGF family at a concentration of 41ng/mL, 42 ng/mL, 43 ng/mL, 44 ng/mL, 45 ng/mL, 46 ng/mL, 47 ng/mL, 48ng/mL or 49 ng/mL. In some embodiments, the Pdx1-positive pancreaticprogenitor cells are contacted with the at least one growth factor fromthe FGF family at a concentration of 51 ng/mL, 52 ng/mL, 53 ng/mL, 54ng/mL, 55 ng/mL, 56 ng/mL, 57 ng/mL, 58 ng/mL or 59 ng/mL. In someembodiments, the Pdx1-positive pancreatic progenitor cells are contactedwith the at least one growth factor from the FGF family at aconcentration of 50 ng/mL.

In some embodiments, the p Pdx1-positive pancreatic progenitor cells arecontacted with the at least one SHH pathway inhibitor at a concentrationof between 0.1 μM and 0.5 μM. In some embodiments, the Pdx1-positivepancreatic progenitor cells are contacted with the at least one SHHpathway inhibitor at a concentration of 0.11 μM, 0.12 μM, 0.13 μM, 0.14μM, 0.15 μM, 0.16 μM, 0.17 μM, 0.18 μM, 0.19 μM, 0.2 μM, 0.21 μM, 0.22μM, 0.23 μM, or 0.24 μM. In some embodiments, the Pdx1-positivepancreatic progenitor cells are contacted with the at least one SHHpathway inhibitor at a concentration of 0.26 μM, 0.27 μM, 0.28 μM, 0.29μM, 0.30 μM, 0.31 μM, 0.32 μM, 0.33 μM, 0.34 μM, 0.35 μM, 0.36 μM, 0.37μM, 0.38 μM, 0.39 μM, 0.40 μM, 0.41 μM, 0.42 μM, 0.43 μM, 0.44 μM, 0.45μM, 0.46 μM, 0.47 μM, 0.48 μM, 0.49 μM. In some embodiments, thePdx1-positive pancreatic progenitor cells are contacted with the atleast one SHH pathway inhibitor at a concentration of 0.25 μM.

In some embodiments, the Pdx1-positive pancreatic progenitor cells arecontacted with the RA signaling pathway activator at a concentration ofbetween 0.01 μM-1.0 μM. In some embodiments, the Pdx1-positivepancreatic progenitor cells are contacted with the RA signaling pathwayactivator at a concentration of 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, 0.06μM, 0.07 μM, 0.08 μM, or 0.09 μM. In some embodiments, the Pdx1-positivepancreatic progenitor cells are contacted with the RA signaling pathwayactivator at a concentration of 0.20 μM, 0.30 μM, 0.40 μM, 0.05 μM, 0.60μM, 0.70 μM, 0.80 μM, or 0.90 μM. In some embodiments, the Pdx1-positivepancreatic progenitor cells are contacted with the RA signaling pathwayactivator at a concentration of 0.1 μM.

In some embodiments, the Pdx1-positive pancreatic progenitor cells arecontacted with the at least one growth factor from the EGF family at aconcentration of between 2 ng/mL-200 ng/mL. In some embodiments, thePdx1-positive pancreatic progenitor cells are contacted with the atleast one growth factor from the EGF family at a concentration of 3ng/mL, 4 ng/mL, 5 ng/mL, 6 ng/mL, 7 ng/mL, 8 ng/mL, 9 ng/mL, 10 ng/mL,11 ng/mL, 12 ng/mL, 13 ng/mL, 14 ng/mL, 16 ng/mL, 17 ng/mL, 18 ng/mL, or19 ng/mL. In some embodiments, the Pdx1-positive pancreatic progenitorcells are contacted with the at least one growth factor from the EGFfamily at a concentration of 30 ng/mL, 35 ng/mL, 40 ng/mL, 45 ng/mL, 50ng/mL, 55 ng/mL, 60 ng/mL, 65 ng/mL, 70 ng/mL, 75 ng/mL, 80 ng/mL, 85ng/mL, 90 ng/mL, 95 ng/mL, or 100 ng/mL. In some embodiments, thePdx1-positive pancreatic progenitor cells are contacted with the atleast one growth factor from the EGF family at a concentration of 21ng/mL, 22 ng/mL, 23 ng/mL, 24 ng/mL, 25 ng/mL, 26 ng/mL, 27 ng/mL, 28ng/mL or 29 ng/mL. In some embodiments, the Pdx1-positive pancreaticprogenitor cells are contacted with the at least one growth factor fromthe EGF family at a concentration of 20 ng/mL.

Generally, the Pdx1-positive pancreatic progenitor cells are maintainedin a suitable culture medium for a period of time sufficient to inducethe differentiation of at least some of the Pdx1-positive pancreaticprogenitor cells in the population into Pdx1-positive, NKX6-1-positivepancreatic progenitor cells. An exemplary suitable culture medium isshown in Table 3 above. In some embodiments, conditions that promotecell clustering comprise a suspension culture. In some embodiments, thesuspension culture is maintained in a spinner flask. In someembodiments, the period of time is at least 5 days. In some embodiments,the suspension culture is replenished every other day. In someembodiments, the β cell-maturation factors are replenished every otherday.

In some embodiments, an activator of protein kinase C is not added tothe suspension culture during the 5 days. In some embodiments, anactivator of protein kinase C is removed from the suspension cultureprior to the 5 days. In some embodiments, the activator of proteinkinase C comprises PdbU. In some embodiments, a BMP signaling pathwayinhibitor is not added to the suspension culture during the 5 days. Insome embodiments, a BMP signaling pathway inhibitor is removed from thesuspension culture prior to the 5 days. In some embodiments, the BMPsignaling pathway inhibitor comprises LDN193189.

In some embodiments, at least 10% of the Pdx1-positive pancreaticprogenitor cells in the population are induced to differentiate intoPdx1-positive, NKX6-1-positive pancreatic progenitor cells. In someembodiments, at least 95% of the Pdx1-positive pancreatic progenitorcells are induced to differentiate into Pdx1-positive, NKX6-1-positivepancreatic progenitor cells.

Generally, any Pdx1-positive pancreatic progenitor cell can bedifferentiated into a Pdx1-positive, NKX6-1-positive pancreaticprogenitor cell. In some embodiments, the NKX6-1-positive pancreaticprogenitor cells express Pdx1, NKX6-1 and/or FoxA2.

In some embodiments, the Pdx1-positive, NKX6-1-positive pancreaticprogenitor cells are obtained by contacting Pdx1-positive pancreaticprogenitor cells under conditions that promote cell clustering with i)at least one growth factor from the FGF family, ii) at least one SHHpathway inhibitor, and optionally iii) low concentrations of a RAsignaling pathway activator, for a period of five days to induce thedifferentiation of at least some of the Pdx1-positive pancreaticprogenitor cells into Pdx1-positive, NKX6-1-positive pancreaticprogenitor cells, wherein the Pdx1-positive, NKX6-1-positive pancreaticprogenitor cells expresses Pdx1 and NKX6-1.

In some embodiments, NKX6-1-positive pancreatic progenitor cells can beobtained by differentiating at least some of the Pdx1-positivepancreatic progenitor cells into Pdx1-positive, NKX6-1-positivepancreatic progenitor cells by a process of contacting the Pdx1-positivepancreatic progenitor cells under conditions that promote cellclustering with i) at least one growth factor from the FGF family, ii)at least one SHH pathway inhibitor, and optionally iii) a RA signalingpathway activator, every other day for a period of five days to inducethe differentiation of at least some of the Pdx1-positive pancreaticprogenitor cells in the population into NKX6-1-positive pancreaticprogenitor cells, wherein the NKX6-1-positive pancreatic progenitorcells expresses Pdx1 and NKX6-1.

In some embodiments, NKX6-1-positive pancreatic progenitor cells can beobtained by differentiating at least some of the Pdx1-positivepancreatic progenitor cells in a population into Pdx1-positive,NKX6-1-positive pancreatic progenitor cells, e.g., by contacting thePdx1-positive pancreatic progenitor cells with i) at least one growthfactor from the FGF family, ii) at least one SHH pathway inhibitor, andoptionally iii) a RA signaling pathway activator, to induce thedifferentiation of at least some of the Pdx1-positive pancreaticprogenitor cells in the population into NKX6-1-positive pancreaticprogenitor cells, wherein the NKX6-1-positive pancreatic progenitorcells expresses Pdx1 and NKX6-1.

Inducing the Differentiation of NKX6-1+ Pancreatic Progenitor Cells toInsulin+ Endocrine Cells

Aspects of the disclosure involve insulin-positive endocrine cells.Insulin-positive endocrine cells of use herein can be derived from anysource or generated in accordance with any suitable protocol. In someaspects, NKX6-1-positive pancreatic progenitor cells are differentiatedto insulin-positive endocrine cells. In some aspects, theinsulin-positive endocrine cells are further differentiated, e.g., byinduction or maturation to SC-β cells.

In some aspects, a method of producing an insulin-positive endocrinecell from an NKX6-1-positive pancreatic progenitor cell comprisescontacting a population of cells (e.g., under conditions that promotecell clustering) comprising NKX6-1-positive pancreatic progenitor cellswith at least two β cell-maturation factors comprising a) a TGF-βsignaling pathway inhibitor, and b) a thyroid hormone signaling pathwayactivator, to induce the differentiation of at least one NKX6-1-positivepancreatic progenitor cell in the population into an insulin-positiveendocrine cell, wherein the insulin-positive pancreatic progenitor cellexpresses insulin.

The disclosure contemplates the use of any TGF-β signaling pathwayinhibitor that induces the differentiation of NKX6-1-positive pancreaticprogenitor cells to differentiate into insulin-positive endocrine cells(e.g., alone, or in combination with other β cell-maturation factors,e.g., a thyroid hormone signaling pathway activator). In someembodiments, the TGF-β signaling pathway comprises TGF-β receptor type Ikinase signaling. In some embodiments, the TGF-β signaling pathwayinhibitor comprises Alk5 inhibitor II.

The disclosure contemplates the use of any thyroid hormone signalingpathway activator that induces the differentiation of NKX6-1-positivepancreatic progenitor cells to differentiate into insulin-positiveendocrine cells (e.g., alone, or in combination with other βcell-maturation factors, e.g., a TGF-β signaling pathway inhibitor). Insome embodiments, the thyroid hormone signaling pathway activatorcomprises triiodothyronine (T3).

In some embodiments, the method comprises contacting the population ofcells (e.g., NKX6-1-positive pancreatic progenitor cells) with at leastone additional β cell-maturation factor. IN some embodiments, the methodcomprises contacting the Pdx1-positive NKX6-1-positive pancreaticprogenitor cells with at least one of i) a SHH pathway inhibitor, ii) aRA signaling pathway activator, iii) a γ-secretase inhibitor, iv) atleast one growth factor from the epidermal growth factor (EGF) family,and optionally v) a protein kinase inhibitor.

In some embodiments, the at least one additional β cell-maturationfactor comprises a γ-secretase inhibitor. The disclosure contemplatesthe use of any γ-secretase inhibitor that is capable of inducing thedifferentiation of NKX6-1-positive pancreatic progenitor cells in apopulation into insulin-positive endocrine cells (e.g., alone, or incombination with any of a TGF-β signaling pathway inhibitor and/or athyroid hormone signaling pathway activator). In some embodiments, theγ-secretase inhibitor comprises XXI. In some embodiments, theγ-secretase inhibitor comprises DAPT.

In some embodiments, the at least one additional β cell-maturationfactor comprises at least one growth factor from the EGF family. Thedisclosure contemplates the use of any growth factor from the EGF familythat is capable of inducing the differentiation of NKX6-1-positivepancreatic progenitor cells in a population into insulin-positiveendocrine cells (e.g., alone, or in combination with any of a TGF-βsignaling pathway inhibitor and/or a thyroid hormone signaling pathwayactivator). In some embodiments, the at least one growth factor from theEGF family comprises betacellulin. In some embodiments, at least onegrowth factor from the EGF family comprises EGF.

In some embodiments, the at least one additional β cell-maturationfactor comprises a low concentration of a retinoic acid (RA) signalingpathway activator. The disclosure contemplates the use of any RAsignaling pathway activator that induces the differentiation ofNKX6-1-positive pancreatic progenitor cells to differentiate intoinsulin-positive endocrine cells (e.g., alone, or in combination withany of a TGF-β signaling pathway inhibitor and/or a thyroid hormonesignaling pathway activator). In some embodiments, the RA signalingpathway activator comprises RA.

In some embodiments, the at least one additional β cell-maturationfactor comprises a sonic hedgehog (SHH) pathway inhibitor. Thedisclosure contemplates the use of any SHH pathway inhibitor thatinduces the differentiation of NKX6-1-positive pancreatic progenitorcells to differentiate into insulin-positive endocrine cells (e.g.,alone, or in combination with any of a TGF-β signaling pathway inhibitorand/or a thyroid hormone signaling pathway activator). In someembodiments, the SHH pathway inhibitor comprises Sant1.

In some embodiments, the population of cells (e.g., NKX6-1-positivepancreatic progenitor cells) is exposed to glucose.

In some embodiments, the population of cells is optionally contactedwith a protein kinase inhibitor. In some embodiments, the population ofcells is not contacted with the protein kinase inhibitor. In someembodiments, the population of cells is contacted with the proteinkinase inhibitor. The disclosure contemplate the use of any proteinkinase inhibitor that is capable of inducing the differentiation ofNKX6-1-positive pancreatic progenitor cells in a population intoinsulin-positive endocrine cells (e.g., alone, or in combination withany of a TGF-β signaling pathway inhibitor and/or a thyroid hormonesignaling pathway activator). In some embodiments, the protein kinaseinhibitor comprises staurosporine.

In some embodiments, the insulin-positive endocrine cells are obtainedby contacting Pdx1-positive, NKX6-1-positive pancreatic progenitor cellswith i) at least one SHH pathway inhibitor, ii) a RA signaling pathwayactivator, iii) a 7-secretase inhibitor, iv) a TGF-β) signaling pathwayinhibitor, v) a TH signaling pathway activator, and vi) at least onegrowth factor from the epidermal growth factor (EGF) family, to inducethe differentiation of at least some of the Pdx1-positive,NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1,insulin-positive endocrine cells, wherein the Pdx1-positive, NKX6-1,insulin-positive endocrine cells express Pdx1, NKX6-1, NKX2-2, Mafb,glis3, Sur, Kir6.2, Znt8, SLC2A1, SLC2A3 and/or insulin.

The skilled artisan will appreciate that the concentrations of agents(e.g., growth factors) employed may vary.

In some embodiments, the NKX6-1-positive pancreatic progenitor cells arecontacted with the at least one TGF-β signaling pathway inhibitor at aconcentration of between 100 nM-100 μM. In some embodiments, theNKX6-1-positive pancreatic progenitor cells are contacted with the atleast one TGF-β signaling pathway inhibitor at a concentration of 10 μM.In some embodiments, the NKX6-1-positive pancreatic progenitor cells arecontacted with the at least one TGF-β signaling pathway inhibitor at aconcentration of 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM,800 nM, or 900 nM. In some embodiments, the NKX6-1-positive pancreaticprogenitor cells are contacted with the at least one TGF-β signalingpathway inhibitor at a concentration of 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7μM, 8 μM, or 9 μM. In some embodiments, the NKX6-1-positive pancreaticprogenitor cells are contacted with the at least one TGF-β signalingpathway inhibitor at a concentration of 9.1 μM, 9.2 μM, 9.3 μM, 9.4 μM,9.5 μM, 9.6 μM, 9.7 μM, 9.8 μM or 9.9 μM. In some embodiments, theNKX6-1-positive pancreatic progenitor cells are contacted with the atleast one TGF-β signaling pathway inhibitor at a concentration of 11 μM,12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, or 19 μM. In someembodiments, the NKX6-1-positive pancreatic progenitor cells arecontacted with the at least one TGF-β signaling pathway inhibitor at aconcentration of 10.1 μM, 10.2 μM, 10.3 μM, 10.4 μM, 10.5 μM, 10.6 μM,10.7 μM, 10.8 μM or 10.9 μM.

In some embodiments, the NKX6-1-positive pancreatic progenitor cells arecontacted with the thyroid hormone signaling pathway activator at aconcentration of between 0.1 μM-10 μM. In some embodiments, theNKX6-1-positive pancreatic progenitor cells are contacted with thethyroid hormone signaling pathway activator at a concentration of 1 μM.In some embodiments, the NKX6-1-positive pancreatic progenitor cells arecontacted with the thyroid hormone signaling pathway activator at aconcentration of 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM,or 0.9 μM. In some embodiments, the NKX6-1-positive pancreaticprogenitor cells are contacted with the γ thyroid hormone signalingpathway activator at a concentration of 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM,1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM or 1.9 μM. In some embodiments, theNKX6-1-positive pancreatic progenitor cells are contacted with thethyroid hormone signaling pathway activator at a concentration of 2 μM,3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, or 9 μM.

In some embodiments, the NKX6-1-positive pancreatic progenitor cells arecontacted with the γ-secretase inhibitor at a concentration of between0.1 μM-10 μM. In some embodiments, the NKX6-1-positive pancreaticprogenitor cells are contacted with the γ-secretase inhibitor at aconcentration of 1 μM. In some embodiments, the NKX6-1-positivepancreatic progenitor cells are contacted with the γ-secretase inhibitorat a concentration of 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM,0.8 μM, or 0.9 μM. In some embodiments, the NKX6-1-positive pancreaticprogenitor cells are contacted with the γ-secretase inhibitor at aconcentration of 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM,1.8 μM or 1.9 μM. In some embodiments, the NKX6-1-positive pancreaticprogenitor cells are contacted with the γ-secretase inhibitor at aconcentration of 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, or 9 μM.

In some embodiments, the NKX6-1-positive pancreatic progenitor cells arecontacted with the at least one growth factor from the EGF family at aconcentration of between 2 ng/mL-200 ng/mL. In some embodiments, theNKX6-1-positive pancreatic progenitor cells are contacted with the atleast one growth factor from the EGF family at a concentration of 3ng/mL, 4 ng/mL, 5 ng/mL, 6 ng/mL, 7 ng/mL, 8 ng/mL, 9 ng/mL, 10 ng/mL,11 ng/mL, 12 ng/mL, 13 ng/mL, 14 ng/mL, 16 ng/mL, 17 ng/mL, 18 ng/mL, or19 ng/mL. In some embodiments, the NKX6-1-positive pancreatic progenitorcells are contacted with the at least one growth factor from the EGFfamily at a concentration of 30 ng/mL, 35 ng/mL, 40 ng/mL, 45 ng/mL, 50ng/mL, 55 ng/mL, 60 ng/mL, 65 ng/mL, 70 ng/mL, 75 ng/mL, 80 ng/mL, 85ng/mL, 90 ng/mL, 95 ng/mL, or 100 ng/mL. In some embodiments, theNKX6-1-positive pancreatic progenitor cells are contacted with the atleast one growth factor from the EGF family at a concentration of 21ng/mL, 22 ng/mL, 23 ng/mL, 24 ng/mL, 25 ng/mL, 26 ng/mL, 27 ng/mL, 28ng/mL or 29 ng/mL. In some embodiments, the NKX6-1-positive pancreaticprogenitor cells are contacted with the at least one growth factor fromthe EGF family at a concentration of 20 ng/mL.

In some embodiments, the NKX6-1-positive pancreatic progenitor cells arecontacted with the RA signaling pathway activator at a concentration ofbetween 0.01 μM-1.0 μM. In some embodiments, the NKX6-1-positivepancreatic progenitor cells are contacted with the RA signaling pathwayactivator at a concentration of 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, 0.06μM, 0.07 μM, 0.08 μM, or 0.09 μM. In some embodiments, theNKX6-1-positive pancreatic progenitor cells are contacted with the RAsignaling pathway activator at a concentration of 0.20 μM, 0.30 μM, 0.40μM, 0.05 μM, 0.60 μM, 0.70 μM, 0.80 μM, or 0.90 μM. In some embodiments,the NKX6-1-positive pancreatic progenitor cells are contacted with theRA signaling pathway activator at a concentration of 0.1 μM.

In some embodiments, the NKX6-1-positive pancreatic progenitor cells arecontacted with a low concentration of a RA signaling pathway activatorat a concentration of between 0.01 μM-1.0 μM. In some embodiments, theNKX6-1-positive pancreatic progenitor cells are contacted with a lowconcentration of the RA signaling pathway activator at a concentrationof 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, 0.06 μM, 0.07 μM, 0.08 μM, or0.09 μM. In some embodiments, the NKX6-1-positive pancreatic progenitorcells are contacted with the low concentration of a RA signaling pathwayactivator at a concentration of 0.20 μM, 0.30 μM, 0.40 μM, 0.05 μM, 0.60μM, 0.70 μM, 0.80 μM, or 0.90 μM. In some embodiments, theNKX6-1-positive pancreatic progenitor cells are contacted with a lowconcentration of the RA signaling pathway activator at a concentrationof 0.1 μM.

In some embodiments, the NKX6-1-positive pancreatic progenitor cells arecontacted with the at least one SHH pathway inhibitor at a concentrationof between 0.1 μM and 0.5 μM. In some embodiments, the NKX6-1-positivepancreatic progenitor cells are contacted with the at least one SHHpathway inhibitor at a concentration of 0.11 μM, 0.12 μM, 0.13 μM, 0.14μM, 0.15 μM, 0.16 μM, 0.17 μM, 0.18 μM, 0.19 μM, 0.2 μM, 0.21 μM, 0.22μM, 0.23 μM, or 0.24 μM. In some embodiments, the NKX6-1-positivepancreatic progenitor cells are contacted with the at least one SHHpathway inhibitor at a concentration of 0.26 μM, 0.27 μM, 0.28 μM, 0.29μM, 0.30 μM, 0.31 μM, 0.32 μM, 0.33 μM, 0.34 μM, 0.35 μM, 0.36 μM, 0.37μM, 0.38 μM, 0.39 μM, 0.40 μM, 0.41 μM, 0.42 μM, 0.43 μM, 0.44 μM, 0.45μM, 0.46 μM, 0.47 μM, 0.48 μM, 0.49 μM. In some embodiments, theNKX6-1-positive pancreatic progenitor cells are contacted with the atleast one SHH pathway inhibitor at a concentration of 0.25 μM.

In some embodiments, the NKX6-1-positive pancreatic progenitor cells arecontacted with the protein kinase inhibitor at a concentration ofbetween 10 nM-1 μM. In some embodiments, the NKX6-1-positive pancreaticprogenitor cells are contacted with the protein kinase inhibitor at aconcentration of 100 nM. In some embodiments, the NKX6-1-positivepancreatic progenitor cells are contacted with the protein kinaseinhibitor at a concentration of 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70nM, 80 nM, or 90 nM. In some embodiments, the NKX6-1-positive pancreaticprogenitor cells are contacted with the protein kinase inhibitor at aconcentration of 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM,180 nM or 190 nM. In some embodiments, the NKX6-1-positive pancreaticprogenitor cells are contacted with the protein kinase inhibitor at aconcentration of 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM,or 900 nM.

In some embodiments, the NKX6-1-positive pancreatic progenitor cells arecontacted with glucose at a concentration of between 1 mM-50 mM. In someembodiments, the NKX6-1-positive pancreatic progenitor cells arecontacted with glucose at a concentration of between 25 mM.

In some embodiments, the insulin-positive endocrine cells can beobtained by differentiating at least some of the Pdx1-positive,NKX6-1-positive pancreatic progenitor cells into Pdx1-positive,NKX6-1-positive, insulin-positive endocrine cells by a process ofcontacting the Pdx1-positive, NKX6-1-positive pancreatic progenitorcells under conditions that promote cell clustering with i) a TGF-βsignaling pathway inhibitor, b) a TH signaling pathway activator, andoptionally c) at least one SHH pathway inhibitor, ii) a RA signalingpathway activator, iii) a γ-secretase inhibitor, and vi) at least onegrowth factor from the epidermal growth factor (EGF) family, every otherday for a period of between five and seven days to induce thedifferentiation of at least some of the Pdx1-positive, NKX6-1-positivepancreatic progenitor cells into Pdx1-positive, NKX6-1, insulin-positiveendocrine cells, wherein the Pdx1-positive, NKX6-1, insulin-positiveendocrine cells express Pdx1, NKX6-1, NKX2-2, Mafb, glis3, Sur1, Kir6.2,Znt8, SLC2A1, SLC2A3 and/or insulin.

In some embodiments, the insulin-positive endocrine cells can beobtained by differentiating at least some of the Pdx1-positive,NKX6-1-positive pancreatic progenitor cells in a population intoPdx1-positive, NKX6-1-positive, insulin-positive endocrine cells, e.g.,by contacting the Pdx1-positive, NKX6-1-positive pancreatic progenitorcells with i) at least one SHH pathway inhibitor, ii) a RA signalingpathway activator, iii) a γ-secretase inhibitor, iv) a TGF-β) signalingpathway inhibitor, v) a TH signaling pathway activator, and vi) at leastone growth factor from the epidermal growth factor (EGF) family, toinduce the differentiation of at least some of the Pdx1-positive,NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1,insulin-positive endocrine cells, wherein the Pdx1-positive, NKX6-1,insulin-positive endocrine cells express Pdx1, NKX6-1, NKX2-2, Mafb,glis3, Sur, Kir6.2, Znt8, SLC2A1, SLC2A3 and/or insulin.

Generally, the population of cells is maintained in a suitable culturemedium for a period of time sufficient to induce the differentiation ofat least one of the NKX6-1-positive pancreatic progenitor cells in thepopulation into an insulin-positive endocrine cell. An exemplary culturemedium is shown in Table 4.

TABLE 4 Agent Concentration MCDB131 1 L Glucose 3.6 g NaHCO3 1.754 gFAF-BSA 20 g ITS-X 5 mL GlutaMAX ™ 10 mL Vitamin C 0.044 g Heparin 10 mgP/S 10 mL

In some embodiments, BE5 media can be used as a suitable culture mediumfor differentiating NKX6-1-positive pancreatic progenitor cells intoinsulin-positive endocrine cells. In some embodiments, a suitableculture medium is shown in Table 5.

In some embodiments, conditions that promote cell clustering comprise asuspension culture. In some embodiments, the period of time comprises aperiod of time sufficient to maximize the number of cells co-expressingC-peptide and Nkx6-1. In some embodiments, the period of time is atleast 5 days. In some embodiments, the period of time is between 5 daysand 7 days. In some embodiments, the period of time is at least 7 days.In some embodiments, the suspension culture is replenished every day(e.g., with β cell-maturation factors). In some embodiments, a period oftime of between 5 days and 7 days maximizes the number of cellsco-expressing C-peptide and Nkx6-1.

In some embodiments, at least 15% of the NKX6-1-positive pancreaticprogenitor cells in the population are induced to differentiate intoinsulin-positive endocrine cells.

In some embodiments, at least 99% of the NKX6-1-positive pancreaticprogenitor cells in the population are induced to differentiate intoinsulin-positive endocrine cells.

Inducing the Maturation of Insulin+ Endocrine Cells into SC-β Cells

Aspects of the disclosure involve SC-β cells. SC-β cells of use hereincan be derived from any source or generated in accordance with anysuitable protocol. In some aspects, insulin-positive endocrine cells areinduced to mature into to SC-β cells.

In some aspects, the disclosure provides a method for generating mature,glucose responsive β cells from insulin-positive endocrine cells, themethod comprising contacting a population of cells (e.g., underconditions that promote cell clustering) comprising insulin-positiveendocrine cells with at least two β cell maturation factors comprisinga) a transforming growth factor-β (TGF-β) signaling pathway inhibitor,b) a thyroid hormone (TH) signaling pathway activator, to induce the invitro maturation of at least one insulin-positive endocrine cell in thepopulation into a SC-β cell.

Aspects of the disclosure involve generating SC-β cells which resembleendogenous mature β cells in form and function, but nevertheless aredistinct from native β cells. The SC-β cells can exhibit a response toat least one glucose challenge. In some embodiments, the SC-β cellsexhibit a response to at least two sequential glucose challenges. Insome embodiments, the SC-β cells exhibit a response to at least threesequential glucose challenges. In some embodiments, the SC-β cellexhibits a response to multiple (e.g., sequential) glucose challengesthat resembles the response of endogenous human islets to multipleglucose challenges. In some embodiments, the SC-β cells are capable ofreleasing or secreting insulin in response to two consecutive glucosechallenges. In some embodiments, the SC-β cells are capable of releasingor secreting insulin in response to three consecutive glucosechallenges. In some embodiments, the SC-β cells are capable of releasingor secreting insulin in response to four consecutive glucose challenges.In some embodiments, the SC-β cells are capable of releasing orsecreting insulin in response to five consecutive glucose challenges. Insome embodiments, the SC-β cells release or secrete insulin in responseto perpetual consecutive glucose challenges. In some embodiments, cellscan be assayed to determine whether they respond to sequential glucosechallenges by determining whether they repeatedly increase intracellularCa²⁺, as described in the examples herein.

In some embodiments, the morphology of the SC-β cells resembles themorphology of endogenous β cells. In some embodiments, the SC-β cellexhibits a glucose stimulated insulin secretion (GSIS) response invitro. In some embodiments, the SC-β cell exhibits a GSIS response invivo. In some embodiments, the SC-β cell exhibits in vitro and in vivoGSIS responses. In some embodiments, the in vitro and/or in vitro GSISresponse resembles the GSIS responses of endogenous mature β cells. Insome embodiments, the SC-β cell exhibits an in vitro (GSIS) responsethat resembles the GSIS response of endogenous β cells. In someembodiments, the SC-β cell exhibits an in vivo GSIS response thatresembles the GSIS response of endogenous β cells. The GSIS response maybe observed immediately upon transplantation into a human or animalsubject. In some embodiments, the GSIS response is observed within twoweeks of transplantation of the SC-β cell into a human or animalsubject. In some embodiments, the GSIS response is observed within twoweeks of transplantation of the SC-β cell into a human or animalsubject. In some embodiments, the GSIS response of the SC-β cell isobserved up to three weeks, four weeks, five weeks, six weeks, sevenweeks, eight weeks, nine weeks, ten weeks, 11 weeks, 12 weeks, 13 weeks,14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 6 months, 7 months, 8months, 9 months, 10 months, 11 months, or up to 1 year or more aftertransplantation of the SC-β cell into a human or animal subject.

In some embodiments, the SC-β cells display at least one marker ofmature endogenous pancreatic β cells. Exemplary markers include, withoutlimitation, Pdx1, HNF6, Ptf1a, Sox9, FoxA2, Nkx2.2, Ngn3, and NKX6-1. Insome embodiments, the expression of a marker selected from the groupconsisting of, HNF6, Ptf1a, Sox9, FoxA2, Nkx2.2, Ngn3, and NKX6-1 isupregulated by a statistically significant amount in the SC-β cellsrelative to the pluripotent stem cells (e.g., embryonic stem cell orinduced pluripotent cell) from which the SC-β cells are derived.

The disclosure contemplates the use of any TGF-β signaling pathwayinhibitor that induces insulin-positive endocrine cells to differentiateand/or mature into SC-β cells (e.g., alone, or with any combination ofat least one thyroid hormone (TH) signaling pathway activator, oroptionally a protein kinase inhibitor). In some embodiments, the TGF-βsignaling pathway comprises TGF-β receptor type I kinase signaling. Insome embodiments, the TGF-β signaling pathway inhibitor comprises Alk5inhibitor II.

The disclosure contemplates the use of any thyroid hormone signalingpathway activator that induces insulin-positive endocrine cells todifferentiate and/or mature into SC-β cells (e.g., alone, or with anycombination of at least one TGF-β signaling pathway inhibitor, oroptionally a protein kinase inhibitor). In some embodiments, the thyroidhormone signaling pathway activator comprises T3.

In some embodiments, the Pdx1-positive, NKX6-1-positive,insulin-positive cells are optionally contacted with a protein kinaseinhibitor. In some embodiments, the Pdx1-positive, NKX6-1-positive,insulin-positive endocrine cells are not contacted with the proteinkinase inhibitor. In some embodiments, the Pdx1-positive,NKX6-1-positive, insulin-positive endocrine cells are contacted with theprotein kinase inhibitor. The disclosure contemplates the use of anyprotein kinase inhibitor that induces insulin-positive endocrine cellsto differentiate and/or mature into SC-β cells (e.g., alone, or with anycombination of at least one TGF-β signaling pathway inhibitor, and/orthyroid hormone signaling pathway activator). In some embodiments, theprotein kinase inhibitor comprises staurosporine.

In some embodiments, the method comprises contacting the population ofcells (e.g., insulin-positive endocrine cells) with at least oneadditional β cell-maturation factor.

In some embodiments, the at least one additional β cell-maturationfactor comprises a cystic fibrosis transmembrane conductance regulator(CFTR) inhibitor. In some embodiments, the method comprises contactingthe Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cellswith a CFTR inhibitor. The disclosure contemplates the use of any CFTRinhibitor that induces insulin-positive endocrine cells to differentiateand/or mature into SC-β cells (e.g., alone, or with any combination ofat least one TGF-β signaling pathway inhibitor, and/or a thyroid hormonesignaling pathway activator, and optionally protein kinase inhibitor).In some embodiments, the CFTR inhibitor comprises Gly-H101.

In some embodiments, the at least one additional β cell-maturationfactor comprises a O-GlcNAcase inhibitor. In some embodiments, themethod comprises contacting the Pdx1-positive, NKX6-1-positive,insulin-positive endocrine cells with a O-GlcNAcase inhibitor. Thedisclosure contemplates the use of any O-GlcNAcase inhibitor thatinduces insulin-positive endocrine cells to differentiate and/or matureinto SC-β cells (e.g., alone, or with any combination of at least oneTGF-β signaling pathway inhibitor, and/or thyroid hormone signalingpathway activator, and optionally a protein kinase inhibitor). In someembodiments, the inhibitor of O-GlcNAcase comprises Thiamet G.

The skilled artisan will appreciate that the concentrations of agents(e.g., growth factors) employed may vary. In some embodiments, theinsulin-positive endocrine cells are contacted with the at least oneTGF-β signaling pathway inhibitor at a concentration of between 100nM-100 μM. In some embodiments, the insulin-positive endocrine cells arecontacted with the at least one TGF-β signaling pathway inhibitor at aconcentration of 10 μM. In some embodiments, the insulin-positiveendocrine cells are contacted with the at least one TGF-β signalingpathway inhibitor at a concentration of 200 nM, 300 nM, 400 nM, 500 nM,600 nM, 700 nM, 800 nM, or 900 nM. In some embodiments, theinsulin-positive endocrine cells are contacted with the at least oneTGF-β signaling pathway inhibitor at a concentration of 2 μM, 3 μM, 4μM, 5 μM, 6 μM, 7 μM, 8 μM, or 9 μM. In some embodiments, theinsulin-positive endocrine cells are contacted with the at least oneTGF-β signaling pathway inhibitor at a concentration of 9.1 μM, 9.2 μM,9.3 μM, 9.4 μM, 9.5 μM, 9.6 μM, 9.7 μM, 9.8 μM or 9.9 μM. In someembodiments, the insulin-positive endocrine cells are contacted with theat least one TGF-β signaling pathway inhibitor at a concentration of 11μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, or 19 μM. In someembodiments, the insulin-positive endocrine cells are contacted with theat least one TGF-β signaling pathway inhibitor at a concentration of10.1 μM, 10.2 μM, 10.3 μM, 10.4 μM, 10.5 μM, 10.6 μM, 10.7 μM, 10.8 μMor 10.9 μM.

In some embodiments, the insulin-positive endocrine cells are contactedwith the thyroid hormone signaling pathway activator at a concentrationof between 0.1 μM-10 μM. In some embodiments, the insulin-positiveendocrine cells are contacted with the thyroid hormone signaling pathwayactivator at a concentration of 1 μM. In some embodiments, theinsulin-positive endocrine cells are contacted with the thyroid hormonesignaling pathway activator at a concentration of 0.2 μM, 0.3 μM, 0.4μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, or 0.9 μM. In some embodiments, theinsulin-positive endocrine cells are contacted with the γ thyroidhormone signaling pathway activator at a concentration of 1.1 μM, 1.2μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM or 1.9 μM. In someembodiments, the insulin-positive endocrine cells are contacted with thethyroid hormone signaling pathway activator at a concentration of 2 μM,3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, or 9 μM.

In some embodiments, the insulin-positive endocrine cells are contactedwith the protein kinase inhibitor at a concentration of between 10 nM-1μM. In some embodiments, the insulin-positive endocrine cells arecontacted with the protein kinase inhibitor at a concentration of 100nM. In some embodiments, the insulin-positive endocrine cells arecontacted with the protein kinase inhibitor at a concentration of 20 nM,30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, or 90 nM. In some embodiments,the insulin-positive endocrine cells are contacted with the proteinkinase inhibitor at a concentration of 110 nM, 120 nM, 130 nM, 140 nM,150 nM, 160 nM, 170 nM, 180 nM or 190 nM. In some embodiments, theinsulin-positive endocrine cells are contacted with the protein kinaseinhibitor at a concentration of 200 nM, 300 nM, 400 nM, 500 nM, 600 nM,700 nM, 800 nM, or 900 nM.

In some embodiments, the Pdx1-positive, NKX6-1-positive,insulin-positive endocrine cells are contacted with the CFTR inhibitorat a concentration of between 100 nM-100 μM. In some embodiments, thePdx1-positive, NKX6-1-positive, insulin-positive endocrine cells arecontacted with the CFTR inhibitor at a concentration of between 10 nM-10μM.

In some embodiments, the Pdx1-positive, NKX6-1-positive,insulin-positive endocrine cells are contacted with the O-GlcNAcaseinhibitor at a concentration of between 100 nM-100 μM. In someembodiments, the Pdx1-positive, NKX6-1-positive, insulin-positiveendocrine cells are contacted with the 0-GlcNAcase inhibitor at aconcentration of 10 nM-10 μM.

In some embodiments, a SC-β cell can be obtained by differentiating atleast some of the Pdx1-positive, NKX6-1-positive, insulin-positiveendocrine cells into SC-β cells by a process of contacting thePdx1-positive, NKX6-1-positive, insulin-positive endocrine cells underconditions that promote cell clustering with i) a transforming growthfactor β (TGF-β) signaling pathway inhibitor, ii) a thyroid hormonesignaling pathway activator, and optionally iii) a protein kinaseinhibitor, every other day for a period of between seven and 14 days toinduce the in vitro maturation of at least some of the Pdx1-positive,NKX6-1-positive, insulin-positive endocrine cells into SC-β cells,wherein the SC-β cells exhibit a GSIS response both in vitro and/or invivo. In some embodiments, the GSIS response resembles the GSIS responseof an endogenous β cell.

In some embodiments, a SC-β cell can be obtained by differentiating atleast some Pdx1-positive, NKX6-1-positive, insulin-positive endocrinecells in a population into SC-β cells, e.g., by contacting thePdx1-positive, NKX6-1-positive, insulin-positive endocrine cells with i)a transforming growth factor β (TGF-β) signaling pathway inhibitor, ii)a thyroid hormone signaling pathway activator, and optionally iii) aprotein kinase inhibitor, to induce the in vitro maturation of at leastsome of the Pdx1-positive, NKX6-1-positive, insulin-producing endocrinecells into SC-β cells, wherein the SC-β cells exhibit a GSIS responseboth in vitro and/or in vivo that resemble the GSIS response of anendogenous β cell.

In some aspects, the disclosure provides a method of generating SC-βcells, the method comprising: contacting Pdx1-positive, NKX6-1-positive,insulin-positive endocrine cells under conditions that promote cellclustering with i) a transforming growth factor β (TGF-β) signalingpathway inhibitor, ii) a thyroid hormone signaling pathway activator,and optionally iii) a protein kinase inhibitor, to induce the in vitromaturation of at least some of the Pdx1-positive, NKX6-1-positive,insulin-positive endocrine cells into SC-β cells, wherein the SC-β cellsexhibit a GSIS response both in vitro and/or in vivo. In someembodiments, the GSIS response resembles the GSIS response of anendogenous β cell.

In some aspects, the disclosure provides a method of generating SC-βcells from pluripotent cells, the method comprising: a) differentiatingpluripotent stem cells in a population into Pdx1-positive pancreaticprogenitor cells; b) differentiating at least some of the Pdx1-positivepancreatic progenitor cells into Pdx1-positive, NKX6-1-positivepancreatic progenitor cells by a process of contacting the Pdx1-positivepancreatic progenitor cells under conditions that promote cellclustering with i) at least one growth factor from the FGF family, ii)at least one SHH pathway inhibitor, and optionally iii) a RA signalingpathway activator, every other day for a period of five days to inducethe differentiation of at least some of the Pdx1-positive pancreaticprogenitor cells in the population into NKX6-1-positive pancreaticprogenitor cells, wherein the NKX6-1-positive pancreatic progenitorcells expresses Pdx1 and NKX6-1; c) differentiating at least some of thePdx1-positive, NKX6-1-positive pancreatic progenitor cells intoPdx1-positive, NKX6-1-positive, insulin-positive endocrine cells by aprocess of contacting the Pdx1-positive, NKX6-1-positive pancreaticprogenitor cells under conditions that promote cell clustering with i) aTGF-β signaling pathway inhibitor, b) a TH signaling pathway activator,and optionally c) at least one SHH pathway inhibitor, ii) a RA signalingpathway activator, iii) a γ-secretase inhibitor, and vi) at least onegrowth factor from the epidermal growth factor (EGF) family, every otherday for a period of between five and seven days to induce thedifferentiation of at least some of the Pdx1-positive, NKX6-1-positivepancreatic progenitor cells into Pdx1-positive, NKX6-1, insulin-positiveendocrine cells, wherein the Pdx1-positive, NKX6-1, insulin-positiveendocrine cells express Pdx1, NKX6-1, NKX2-2, Mafb, glis3, Sur, Kir6.2,Znt8, SLC2A1, SLC2A3 and/or insulin; and d) differentiating at leastsome of the Pdx1-positive, NKX6-1-positive, insulin-positive endocrinecells into SC-β cells by a process of contacting the Pdx1-positive,NKX6-1-positive, insulin-positive endocrine cells under conditions thatpromote cell clustering with i) a transforming growth factor β (TGF-β)signaling pathway inhibitor, ii) a thyroid hormone signaling pathwayactivator, and optionally iii) a protein kinase inhibitor, every otherday for a period of between seven and 14 days to induce the in vitromaturation of at least some of the Pdx1-positive, NKX6-1-positive,insulin-positive endocrine cells into SC-β cells, wherein the SC-β cellsexhibit a GSIS response in vitro and/or in vivo. In some embodiments,the GSIS response resembles the GSIS response of an endogenous mature βcells.

In some aspects, the disclosure provides a method of generating SC-βcells from pluripotent cells, the method comprising: a) differentiatingat least some pluripotent cells in a population into Pdx1-positivepancreatic progenitor cells; b) differentiating at least some of thePdx1-positive pancreatic progenitor cells into Pdx1-positive,NKX6-1-positive pancreatic progenitor cells by a process of contactingthe Pdx1-positive pancreatic progenitor cells under conditions thatpromote cell clustering with i) KGF, ii) Sant1, and optionally iii) lowconcentrations of RA, every other day for a period of five days toinduce the differentiation of at least one Pdx1-positive pancreaticprogenitor cell in the population into NKX6-1-positive pancreaticprogenitor cells, wherein the NKX6-1-positive pancreatic progenitorcells expresses Pdx1 and NKX6-1; c) differentiating at least some of thePdx1-positive, NKX6-1-positive pancreatic progenitor cells intoPdx1-positive, NKX6-1-positive, insulin-positive endocrine cells by aprocess of contacting the Pdx1-positive, NKX6-1-positive pancreaticprogenitor cells with i) Alk5 Inhibitor II, ii) T3, and optionally iii)Sant1, iv) RA, v) XXI, and vi) betacellulin, every other day for aperiod of between five and seven days to induce the differentiation ofat least some of the Pdx1-positive, NKX6-1-positive pancreaticprogenitor cells into Pdx1-positive, NKX6-1, insulin-positive endocrinecells, wherein the Pdx1-positive, NKX6-1, insulin-positive endocrinecells express Pdx1, NKX6-1, NKX2-2, Mafb, glis3, Sur, Kir6.2, Znt8,SLC2A1, SLC2A3 and/or insulin; and d) differentiating at least some ofthe Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cellsinto SC-β cells by a process of contacting the Pdx1-positive,NKX6-1-positive, insulin-positive endocrine cells under conditions thatpromote cell clustering with i) Alk5 inhibitor II, ii) T3, andoptionally iii) staurosporine, every other day for a period of betweenseven and 14 days to induce the in vitro maturation of at least some ofthe Pdx1-positive, NKX6-1-positive, insulin-producing endocrine cellsinto SC-β cells, wherein the SC-β cells exhibit a GSIS response in vitroand in vivo that resemble the GSIS response of an endogenous β cell.

Generally, the Pdx1-positive, NKX6-1-positive, insulin-positiveendocrine cells are maintained in a suitable culture medium for a periodof time sufficient to induce the in vitro maturation of at least some ofthe Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cellsinto SC-β cells. Exemplary suitable culture media are shown above inTable 4 and below in Table 5.

TABLE 5 CMRL Islet Media (“CMRLS”) CMRL 1066 Supplemented cat#99-603-CVMediatech We supplement with 10% Hyclone FBS

In some embodiments, the suitable culture medium comprises ConnoughtMedical Research Laboratories 1066 supplemented islet media (CMRLS). Insome embodiments, the suitable culture medium comprises a component ofCMRLS (e.g., supplemental zinc). In some embodiments, the suitableculture medium is shown in Table 3. In some embodiments, the CMRLS issupplemented with serum (e.g., human). In some embodiments, the CMRLS issupplemented with serum replacements (e.g., KOSR). In some embodiments,the CMRLS is supplemented with fetal bovine serum. In some embodiments,the CMRLS is supplemented with 10% fetal bovine serum. In someembodiments, a suitable culture medium for differentiatinginsulin-positive endocrine cells into SC-β cells comprises S3 media. Insome embodiments, conditions that promote cell clustering comprise asuspension culture. In some embodiments, the period of time comprises atleast 7 days. In some embodiments, the period of time comprises between7 days and 21 days. In some embodiments, the period of time comprisesbetween 7 and 14 days. In some embodiments, the period of time comprisesbetween 10 and 14 days. In some embodiments, the period of timecomprises 14 days. In some embodiments, the suspension culture isreplenished every other day (e.g., with the β cell-maturation factors).

In some embodiments, at least 1%, at least 2%, at least 3%, at least 4%,at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, atleast 10%, at least 20%, at least 30%, at least 40%, at least 50% of thePdx1-positive, NKX6-1-positive, insulin-positive endocrine cells areinduced to mature into SC-β cells. In some embodiments, at least atleast 60%, at least 70%, at least 80%, at least 90%, at least 99% of thePdx1-positive, NKX6-1-positive, insulin-positive endocrine cells areinduced to mature into SC-β cells. In some embodiments, at least 30% ofthe cells generated comprise SC-β cells. In some embodiments, the SC-βcells express C-peptide, insulin, NKX6-1, Pdx1, and co-express NKX6-1and C-peptide.

In some embodiments, the SC-β cells comprise human cells. In someembodiments, generation of the SC-β cells in vitro is scalable.

Isolated Populations of Cells

Aspects of the disclosure relate to isolated populations of cellsproduced according to a method described herein. In some embodiments, apopulation of SC-β cells are produced by contacting at least oneinsulin-positive endocrine cell or precursor thereof with at least one βcell maturation factors described herein. In some embodiments, apopulation of SC-β cells are produced by contacting at least oneinsulin-positive endocrine cell or precursor thereof with at least two βcell maturation factors described herein. In some embodiments, apopulation of SC-β cells are produced by contacting at least oneinsulin-positive endocrine cell or precursor thereof with at least threeβ cell maturation factors described herein. In some embodiments, apopulation of SC-β cells are produced by contacting at least oneinsulin-positive endocrine cell or precursor thereof with at least fourβ cell maturation factors described herein. In some embodiments, apopulation of SC-β cells are produced by contacting at least oneinsulin-positive endocrine cell or precursor thereof with at least fiveβ cell maturation factors described herein. In some embodiments, apopulation of SC-β cells are produced by contacting at least oneinsulin-positive endocrine cell or precursor thereof with at least six,at least seven, at least eight, at least nine, or at least ten β cellmaturation factors described herein.

In some aspects, the disclosure provides an isolated population ofdefinitive endoderm cells. An isolated population of definitive endodermcells can be obtained by differentiating at least some pluripotent cellsin a population into definitive endoderm cells, e.g., by a process ofcontacting a population of pluripotent cells with i) at least one growthfactor from the TGF-β superfamily, and ii) a Wnt signaling pathwayactivator, to induce the differentiation of at least some of thepluripotent cells in the population into definitive endoderm cells,wherein the definitive endoderm cells express at least one markercharacteristic of definitive endoderm.

In some aspects, the disclosure provides an isolated population ofprimitive gut tube cells. An isolated population of primitive gut tubecells can be obtained by differentiating at least some definitiveendoderm cells in a population into primitive gut tube cells, e.g., by aprocess of contacting the definitive endoderm cells with at least onegrowth factor from the fibroblast growth factor (FGF) family, to inducethe differentiation of at least some of the definitive endoderm cellsinto primitive gut tube cells, wherein the primitive gut tube cellsexpress at least one marker characteristic of definitive endoderm.

In some aspects, the disclosure provides an isolated population ofPdx1-positive pancreatic progenitor cells. An isolated population ofPdx1-positive pancreatic progenitor cells can be obtained bydifferentiating at least some primitive gut tube cells in a populationinto Pdx1-positive pancreatic progenitor cells, e.g., by a process ofcontacting the primitive gut tube cells with i) at least one bonemorphogenic protein (BMP) signaling pathway inhibitor, ii) at least onegrowth factor from the FGF family, iii) at least one SHH pathwayinhibitor, iv) at least one retinoic acid (RA) signaling pathwayactivator; and v) at least one protein kinase C activator, to induce thedifferentiation of at least some of the primitive gut tube cells intoPdx1-positive pancreatic progenitor cells, wherein the Pdx1-positivepancreatic progenitor cells express Pdx1.

In some aspects, the disclosure provides an isolated population ofNKX6-1-positive pancreatic progenitor cells. An isolated population ofNKX6-1-positive pancreatic progenitor cells can be obtained bydifferentiating at least some Pdx1-positive pancreatic progenitor cellsin a population into Pdx1-positive, NKX6-1-positive pancreaticprogenitor cells, e.g., by a process of contacting the Pdx1-positivepancreatic progenitor cells with i) at least one growth factor from theFGF family, ii) at least one SHH pathway inhibitor, and optionally iii)a RA signaling pathway activator, to induce the differentiation of atleast one Pdx1-positive pancreatic progenitor cell in the populationinto NKX6-1-positive pancreatic progenitor cells, wherein theNKX6-1-positive pancreatic progenitor cells expresses Pdx1 and NKX6-1.

In some aspects, the disclosure provides an isolated population ofinsulin-positive endocrine cells. An isolated population ofinsulin-positive endocrine cells can be obtained by differentiating atleast some Pdx1-positive, NKX6-1-positive pancreatic progenitor cells ina population into Pdx1-positive, NKX6-1-positive, insulin-positiveendocrine cells, e.g., by a process of contacting the Pdx1-positive,NKX6-1-positive pancreatic progenitor cells with i) a TGF-β) signalingpathway inhibitor, ii) a TH signaling pathway activator, and optionallyat least one additional β cell-maturation factor selected from the groupconsisting of i) at least one SHH pathway inhibitor, ii) a RA signalingpathway activator, iii) a γ-secretase inhibitor, iv) and vi) at leastone growth factor from the epidermal growth factor (EGF) family, toinduce the differentiation of at least some of the Pdx1-positive,NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1,insulin-positive endocrine cells, wherein the Pdx1-positive, NKX6-1,insulin-positive endocrine cells express Pdx1, NKX6-1, NKX2-2, Mafb,glis3, Sur, Kir6.2, Znt8, SLC2A1, SLC2A3 and/or insulin.

In some aspects, the disclosure provides an isolated population of SC-βcells. An isolated population of SC-β cells can be obtained bydifferentiating at least some Pdx1-positive, NKX6-1-positive,insulin-positive endocrine cells in a population into SC-β cells, e.g.,by a process of contacting the Pdx1-positive, NKX6-1-positive,insulin-positive endocrine cells with i) a transforming growth factor β(TGF-0) signaling pathway inhibitor, ii) a thyroid hormone signalingpathway activator, and optionally iii) a protein kinase inhibitor, toinduce the in vitro maturation of at least some of the Pdx1-positive,NKX6-1-positive, insulin-positive endocrine cells into SC-β cells,wherein the SC-β cells exhibit a GSIS response in vitro and/or in vivo.In some embodiments, the GSIS response resembles the GSIS response of anendogenous β cell.

Aspects of the disclosure involve microcapsules comprising isolatedpopulations of cells described herein (e.g., SC-β cells). Microcapsulesare well known in the art. Suitable examples of microcapsules aredescribed in the literature (e.g., Jahansouz et al., “Evolution ofβ-Cell Replacement Therapy in Diabetes Mellitus: Islet CellTransplantation” Journal of Transplantation 2011; Volume 2011, ArticleID 247959; Orive et al., “Application of cell encapsulation forcontrolled delivery of biological therapeutics”, Advanced Drug DeliveryReviews (2013), available on the worldwide web at subdomaindx.doi.org/10.1016/jaddr.2013.07.009; Hernandez et al., “Microcapsulesand microcarriers for in situ cell delivery”, Advanced Drug DeliveryReviews 2010; 62:711-730; Murua et al., “Cell microencapsulationtechnology: Towards clinical application”, Journal of Controlled Release2008; 132:76-83; and Zanin et al., “The development of encapsulated celltechnologies as therapies for neurological and sensory diseases”,Journal of Controlled Release 2012; 160:3-13). Microcapsules can beformulated in a variety of ways. Exemplary microcapsules comprise analginate core surrounded by a polycation layer covered by an outeralignate membrane. The polycation membrane forms a semipermeablemembrane, which imparts stability and biocompatibility. Examples ofpolycations include, without limitation, poly-L-lysine,poly-L-ornithine, chitosan, lactose modified chitosan, andphotopolymerized biomaterials. In some embodiments, the alginate core ismodified, for example, to produce a scaffold comprising an alginate corehaving covalently conjugated oligopeptides with an RGD sequence(arginine, glycine, aspartic acid). In some embodiments, the alginatecore is modified, for example, to produce a covalently reinforcedmicrocapsule having a chemoenzymatically engineered alginate of enhancedstability. In some embodiments, the alginate core is modified, forexample, to produce membrane-mimetic films assembled by in-situpolymerization of acrylate functionalized phospholipids. In someembodiments, microcapsules are composed of enzymatically modifiedalginates using epimerases. In some embodiments, microcapsules comprisecovalent links between adjacent layers of the microcapsule membrane. Insome embodiment, the microcapsule comprises a subsieve-size capsulecomprising aliginate coupled with phenol moieties. In some embodiments,the microcapsule comprises a scaffold comprising alginate-agarose. Insome embodiments, the SC-β cell is modified with PEG before beingencapsulated within alginate. In some embodiments, the isolatedpopulations of cells, e.g., SC-β cells are encapsulated in photoreactiveliposomes and alginate. It should be appreciate that the alginateemployed in the microcapsules can be replaced with other suitablebiomaterials, including, without limitation, PEG, chitosan, PES hollowfibers, collagen, hyaluronic acid, dextran with RGD, EHD and PEGDA, PMBVand PVA, PGSAS, agarose, agarose with gelatin, PLGA, and multilayerembodiments of these.

In some embodiments, compositions comprising populations of cellsproduced according to the methods described herein can also be used asthe functional component in a mechanical device designed to produce oneor more of the endocrine polypeptides of pancreatic islet cells. In itssimplest form, the device contains a population of pancreatic beta cells(e.g., produced from populations of insulin-positive endocrine cells orprecursors thereof) behind a semipermeable membrane that preventspassage of the cell population, retaining them in the device, butpermits passage of insulin, glucagon, or somatostatin secreted by thecell population. This includes populations of pancreatic beta cells thatare microencapsulated, typically in the form of cell clusters to permitthe cell interaction that inhibits dedifferentiation. For example, U.S.Pat. No. 4,391,909 describe islet cells encapsulated in a spheroidsemipermeable membrane made up of polysaccharide polymers >3,000 mol.wt. that are cross-linked so that it is permeable to proteins the sizeof insulin, but impermeable to molecules over 100,000 mol. wt. U.S. Pat.No. 6,023,009 describes islet cells encapsulated in a semipermeablemembrane made of agarose and agaropectin. Microcapsules of this natureare adapted for administration into the body cavity of a diabeticpatient, and are thought to have certain advantages in reducinghistocompatibility problems or susceptibility to bacteria.

More elaborate devices are also contemplated for use to comprise apopulation of pancreatic beta cells produced from insulin-positiveendocrine cells or precursors thereof according to the methods describedherein, either for implantation into diabetic patients, or forextracorporeal therapy. U.S. Pat. No. 4,378,016 describes an artificialendocrine gland containing an extracorporeal segment, a subcutaneoussegment, and a replaceable envelope containing the hormone-producingcells. U.S. Pat. No. 5,674,289 describes a bioartificial pancreas havingan islet chamber, separated by a semipermeable membrane to one or morevascularizing chambers open to surrounding tissue. Useful devicestypically have a chamber adapted to contain the islet cells, and achamber separated from the islet cells by a semipermeable membrane whichcollects the secreted proteins from the islet cells, and which may alsopermit signaling back to the islet cells, for example, of thecirculating glucose level.

Aspects of the disclosure involve assays comprising isolated populationsof cells described herein (e.g., SC-β cells). In some embodiments, theassays can be used for identifying one or more candidate agents whichpromote or inhibit a cell fate selected from the group consisting of βcell proliferation, β cell replication, and β cell death, β cellfunction, β cell susceptibility to immune attack, or β cellsusceptibility to dedifferentiation or differentiation. In someembodiments, the assays can be used for identifying one or morecandidate agents which promote the differentiation of at least oneinsulin-positive endocrine cell or a precursor thereof into SC-β cells.In some embodiments, the assays can be used for identifying one or morecandidate agents which stimulate β cells to produce insulin or increaseproduction or secretion of insulin.

The disclosure contemplates methods in which SC-β cells are generatedaccording to the methods described herein from iPS cells derived fromcells extracted or isolated from individuals suffering from a disease(e.g., diabetes, obesity, or a β cell-related disorder), and those SC-βcells are compared to normal β cells from healthy individuals not havingthe disease to identify differences between the SC-β cells and normal βcells which could be useful as markers for disease (e.g., epigeneticand/or genetic). In some embodiments, β cells are obtained from adiabetic individual and compared to normal β cells, and then the β cellsare reprogrammed to iPS cells and the iPS cells are analyzed for geneticand/or epigenetic markers which are present in the β cells obtained fromthe diabetic individual but not present in the normal β cells, toidentify markers (e.g., pre-diabetic). In some embodiments, the iPScells and/or SC-β derived from diabetic patients are used to screen foragents (e.g., agents which are able to modulate genes contributing to adiabetic phenotype).

Methods of Differentiation of Insulin-Positive Endocrine Cells to SC-βCells

Generating SC-β cells by conversion of at least one insulin-positiveendocrine cell or a precursor thereof using the methods of thedisclosure has a number of advantages. First, the methods of thedisclosure allow one to generate autologous SC-β cells, which are cellsspecific to and genetically matched with an individual. In general,autologous cells are less likely than non-autologous cells to be subjectto immunological rejection. The cells are derived from at least oneinsulin-positive endocrine cell or a precursor thereof, e.g., apancreatic progenitor obtained by reprogramming a somatic cell (e.g., afibroblast) from the individual to an induced pluripotent state, andthen culturing the pluripotent cells to differentiate at least some ofthe pluripotent cells to at least one insulin-positive endocrine cell ora precursor thereof, followed by transplantation of the at least oneinsulin-positive endocrine cell or precursor thereof into the individualsuch that the at least one insulin-positive endocrine cell or precursorthereof matures in vivo into a SC-β cell, or induced maturation in vitroof the at least one insulin-positive endocrine cell into a SC-β cell.

In some embodiments, a subject from which at least one insulin-positiveendocrine cell or precursor thereof are obtained is a mammalian subject,such as a human subject. In some embodiments, the subject is sufferingfrom a cell disorder. In some embodiments, the subject is suffering fromdiabetes. In some embodiments, the subject is suffering fromprediabetes. In such embodiments, the at least one insulin-positiveendocrine cell or precursor thereof can be differentiated into a SC-βcell ex vivo by the methods as described herein and then administered tothe subject from which the cells were harvested in a method to treat thesubject for the β cell disorder (e.g., diabetes).

In some embodiments, at least one insulin-positive endocrine cell or aprecursor thereof is located within a subject (in vivo) and is convertedto become a SC-β cell by the methods as disclosed herein in vivo. Insome embodiments, conversion of at least one insulin-positive endocrinecell or a precursor thereof to a SC-β cell in vivo can be achieved byadministering to a subject a composition comprising at least one, atleast two, at least three, at least four, at least five, or at leastsix, or more β cell maturation factors as described herein. In someembodiments, conversion of at least one insulin-positive endocrine cellor a precursor thereof to a SC-β cell in vivo can be achieved byadministering to a subject a composition comprising at least one, atleast two, at least three, at least four, at least five, or at least sixβ cell maturation factors as described herein.

In some embodiments, contacting may be performed by maintaining the atleast one insulin-positive endocrine cell or a precursor thereof inculture medium comprising the one or more β cell maturation factors. Insome embodiments at least one insulin-positive endocrine cell or aprecursor thereof can be genetically engineered. In some embodiments, atleast one insulin-positive endocrine cell or a precursor thereof can begenetically engineered to express one or more β cell markers asdisclosed herein, for example express at least one a polypeptideselected from pancreatic and duodenal homeobox 1 (PDX-1) polypeptide,insulin, c-peptide, amylin, E-cadherin, Hnf3p, PCI/3, B2, Nkx2.2,NKX6-1, GLUT2, PC2, ZnT-8, or an amino acid sequences substantiallyhomologous thereof, or functional fragments or functional variantsthereof.

Where the at least one insulin-positive endocrine cell or a precursorthereof is maintained under in vitro conditions, conventional tissueculture conditions and methods can be used, and are known to those ofskill in the art. Isolation and culture methods for various cells arewell within the abilities of one skilled in the art.

In the methods of the disclosure at least one insulin-positive endocrinecell or a precursor thereof can, in general, be cultured under standardconditions of temperature, pH, and other environmental conditions, e.g.,as adherent cells in tissue culture plates at 37° C. in an atmospherecontaining 5-10% C02. The cells and/or the culture medium areappropriately modified to achieve conversion to SC-β cells as describedherein. In certain embodiments, at least one insulin-positive endocrinecell or a precursor thereof, e.g., a pancreatic progenitor can becultured on or in the presence of a material that mimics one or morefeatures of the extracellular matrix or comprises one or moreextracellular matrix or basement membrane components. In someembodiments MATRIGEL™ is used. Other materials include proteins ormixtures thereof such as gelatin, collagen, fibronectin, etc. In certainembodiments of the invention, at least one insulin-positive endocrinecell or a precursor thereof can be cultured in the presence of a feederlayer of cells. Such cells may, for example, be of murine or humanorigin. They can also be irradiated, chemically inactivated by treatmentwith a chemical inactivator such as mitomycin c, or otherwise treated toinhibit their proliferation if desired. In other embodiments at leastone insulin-positive endocrine cell or a precursor thereof are culturedwithout feeder cells. In some embodiments, the insulin-positiveendocrine cells or precursors thereof are cultured in conditions thatpromote cell clustering. As used herein, “conditions that promote cellclustering” refers to any condition which stimulates the clustering ofcells during differentiation of the cells toward SC-β cells. In someembodiments, conditions that promote cell clustering comprise asuspension culture. Boretti and Gooch (Tissue Eng. 2006 April;12(4):939-48) report that culture in least adherent conditions(low-serum medium, low-adherent substrate) stimulated cell clustering inthe transdifferentiation of adult pancreatic ductal epithelial cells tobeta cells in vitro. Accordingly, without wishing to be bound by theory,in some embodiments, conditions that promote cell clustering compriseminimally adherent conditions, e.g., low-serum medium, low-adherentsubstrate.

In certain examples, the β cell maturation factors can be used to inducethe differentiation of at least one insulin-positive endocrine cell orprecursor thereof by exposing or contacting at least oneinsulin-positive endocrine cell or precursor thereof with an effectiveamount of a β cell maturation factor described herein to differentiatethe at least one insulin-positive endocrine cell or precursor thereofinto at least one SC-β cell (e.g., a mature pancreatic β cell).

Accordingly, included herein are cells and compositions made by themethods described herein. The exact amount and type of β cell maturationfactor can vary depending on the number of insulin-positive endocrinecells or precursors thereof, the desired differentiation stage and thenumber of prior differentiation stages that have been performed.

In certain examples, a β cell maturation factor is present in aneffective amount. As used herein, “effective amount” refers to theamount of the compound that should be present for the differentiation ofat least 10% or at least 20% or at least 30% of the cells in apopulation of insulin-positive endocrine cells or precursors thereofinto SC-β cells.

In additional examples, β cell maturation factors can be present in theculture medium of the at least one insulin-positive endocrine cell orprecursor thereof, or alternatively, the β cell maturation factors maybe added to the at least one insulin-positive endocrine cell orprecursor thereof during some stage of growth.

Confirmation of the Presence and the Identification of Cells SC-β Cells

One can use any means common to one of ordinary skill in the art toconfirm the presence of a SC-β cell, e.g. a mature pancreatic β cellproduced the induction of the differentiation of at least oneinsulin-positive endocrine cell or precursor thereof by exposure to atleast one β cell maturation factor as described herein.

In some embodiments, the presence of β cell markers, e.g. chemicallyinduced SC-β cells, can be done by detecting the presence or absence ofone or more markers indicative of an endogenous β cell. In someembodiments, the method can include detecting the positive expression(e.g. the presence) of a marker for mature β cells. In some embodiments,the marker can be detected using a reagent, e.g., a reagent for thedetection of NKX6-1 and C-peptide. In particular, SC-β cells hereinexpress NKX6-1 and C-peptide, and do not express significant levels ofother markers which would be indicative of immature β cells (e.g.,MafB). A reagent for a marker can be, for example, an antibody againstthe marker or primers for a RT-PCR or PCR reaction, e.g., asemi-quantitative or quantitative RT-PCR or PCR reaction. Such markerscan be used to evaluate whether a SC-β cell has been produced. Theantibody or other detection reagent can be linked to a label, e.g., aradiological, fluorescent (e.g., GFP) or colorimetric label for use indetection. If the detection reagent is a primer, it can be supplied indry preparation, e.g., lyophilized, or in a solution.

The progression of at least one insulin-positive endocrine cell orprecursor thereof to a SC-β cell can be monitored by determining theexpression of markers characteristic of mature β cells. In someprocesses, the expression of certain markers is determined by detectingthe presence or absence of the marker. Alternatively, the expression ofcertain markers can be determined by measuring the level at which themarker is present in the cells of the cell culture or cell population.In certain processes, the expression of markers characteristic of SC-βcells as well as the lack of significant expression of markerscharacteristic of the insulin-positive endocrine cells or precursorsthereof, e.g., pluripotent stem cell or pancreatic progenitor cell fromwhich it was derived is determined.

As described in connection with monitoring the production of a SC-β cell(e.g., a mature pancreatic β cell) from an insulin-positive endocrinecell, qualitative or semi-quantitative techniques, such as blot transfermethods and immunocytochemistry, can be used to measure markerexpression, using methods commonly known to persons of ordinary skill inthe art. Alternatively, marker expression can be accurately quantitatedthrough the use of technique such as quantitative-PCR by methodsordinarily known in the art. Additionally, it will be appreciated thatat the polypeptide level, many of the markers of pancreatic islethormone-expressing cells are secreted proteins. As such, techniques formeasuring extracellular marker content, such as ELISA, may be utilized.

SC-β cells can also be characterized by the down-regulation of markerscharacteristic of the pluripotent stem from which the SC-β cell isinduced from. For example, SC-β cells derived from pluripotent stem cellmay be characterized by a statistically significant down-regulation ofthe pluripotent stem cell markers alkaline phosphatase (AP), NANOG,OCT-4, SOX-2, SSEA4, TRA-1-60 or TRA-1-81 in the mature relative to theexpression in the pluripotent stem cell from which it was derived. Othermarkers expressed by pluripotent cell markers, include but are notlimited to alkaline phosphatase (AP); ABCG2; stage specific embryonicantigen-1 (SSEA-1); SSEA-3; SSEA-4; TRA-1-60; TRA-1-81; Tra-2-49/6E;ERas/ECATS, E-cadherin; βIII-tubulin; α-smooth muscle actin (α-SMA);fibroblast growth factor 4 (Fgf4), Cripto, Dax1; zinc finger protein 296(Zfp296); N-acetyltransferase-1 (Nat1); (ES cell associated transcript 1(ECAT1); ESG1/DPPAS/ECAT2; ECAT3; ECAT6; ECAT7; ECAT8; ECAT9; ECAT10;ECAT15-1; ECAT15-2; Fth117; Sa114; undifferentiated embryonic celltranscription factor (Utf1); Rex1; p53; G3PDH; telomerase, includingTERT; silent X chromosome genes; Dnmt3a; Dnmt3b; TRIM28; F-boxcontaining protein 15 (Fbx15); Nanog/ECAT4; Oct3/4; Sox2; Klf4; c-Myc;Esrrb; TDGF1; GABRB3; Zfp42, FoxD3; GDF3; CYP25A1; developmentalpluripotency-associated 2 (DPPA2); T-cell lymphoma breakpoint 1 (Tcl1);DPPA3/Stella; DPPA4; Dnmt3L; Sox15; Stat3; Grb2; SV40 Large T Antigen;HPV16 E6; HPV16 E7, β-catenin, and Bmi1 and other general markers forpluripotency, etc, and at least one or more of these are down regulatedby a statistically significant amount in a mature as compared to thepluripotent stem cell from which they were derived.

It is understood that the present invention is not limited to thosemarkers listed as mature β cell markers herein, and the presentinvention also encompasses markers such as cell surface markers,antigens, and other gene products including ESTs, RNA (includingmicroRNAs and antisense RNA), DNA (including genes and cDNAs), andportions thereof.

Enrichment, Isolation and Purification of a SC-β Cell

Another aspect of the present invention relates to the isolation of apopulation of SC-β cells from a heterogeneous population of cells, sucha mixed population of cells comprising SC-β cells and insulin-positiveendocrine cells or precursors thereof from which the SC-β cells werederived. A population of SC-β cells produced by any of theabove-described processes can be enriched, isolated and/or purified byusing any cell surface marker present on the SC-β cells which is notpresent on the insulin-positive endocrine cell or precursor thereof fromwhich it was derived. Such cell surface markers are also referred to asan affinity tag which is specific for a SC-β cell. Examples of affinitytags specific for SC-β cells are antibodies, ligands or other bindingagents that are specific to a marker molecule, such as a polypeptide,that is present on the cell surface of a SC-β cells but which is notsubstantially present on other cell types (e.g. insulin-positiveendocrine cells or precursors thereof). In some processes, an antibodywhich binds to a cell surface antigen on a SC-β cell (e.g. a human SC-βcell) is used as an affinity tag for the enrichment, isolation orpurification of chemically induced (e.g. by contacting with at least oneβ cell maturation factor as described herein) SC-β cells produced by themethods described herein. Such antibodies are known and commerciallyavailable.

The skilled artisan will readily appreciate the processes for usingantibodies for the enrichment, isolation and/or purification of SC-βcell. For example, in some embodiments, the reagent, such as anantibody, is incubated with a cell population comprising SC-β cells,wherein the cell population has been treated to reduce intercellular andsubstrate adhesion. The cell population are then washed, centrifuged andresuspended. In some embodiments, if the antibody is not already labeledwith a label, the cell suspension is then incubated with a secondaryantibody, such as an FITC-conjugated antibody that is capable of bindingto the primary antibody. The SC-β cells are then washed, centrifuged andresuspended in buffer. The SC-β cell suspension is then analyzed andsorted using a fluorescence activated cell sorter (FACS).Antibody-bound, fluorescent reprogrammed cells are collected separatelyfrom non-bound, non-fluorescent cells (e.g. immature, insulin-producingcells), thereby resulting in the isolation of SC-β cells from othercells present in the cell suspension, e.g. insulin-positive endocrinecells or precursors thereof, or immature, insulin-producing cell (e.g.other differentiated cell types).

In another embodiment of the processes described herein, the isolatedcell composition comprising SC-β cells can be further purified by usingan alternate affinity-based method or by additional rounds of sortingusing the same or different markers that are specific for SC-β cells.For example, in some embodiments, FACS sorting is used to first isolatea SC-β cell which expresses NKX6-1, either alone or with the expressionof C-peptide, or alternatively with a β cell marker disclosed hereinfrom cells that do not express one of those markers (e.g. negativecells) in the cell population. A second FAC sorting, e.g. sorting thepositive cells again using FACS to isolate cells that are positive for adifferent marker than the first sort enriches the cell population forreprogrammed cells.

In an alternative embodiment, FACS sorting is used to separate cells bynegatively sorting for a marker that is present on most insulin-positiveendocrine cells or precursors thereof but is not present on SC-β cells.

In some embodiments of the processes described herein, SC-β cells arefluorescently labeled without the use of an antibody then isolated fromnon-labeled cells by using a fluorescence activated cell sorter (FACS).In such embodiments, a nucleic acid encoding GFP, YFP or another nucleicacid encoding an expressible fluorescent marker gene, such as the geneencoding luciferase, is used to label reprogrammed cells using themethods described above. For example, in some embodiments, at least onecopy of a nucleic acid encoding GFP or a biologically active fragmentthereof is introduced into at least one insulin-positive endocrine cellwhich is first chemically induced into a SC-β cell, where a downstreamof a promoter expressed in SC-β cell, such as the insulin promoter, suchthat the expression of the GFP gene product or biologically activefragment thereof is under control of the insulin promoter.

In addition to the procedures just described, chemically induced SC-βcells may also be isolated by other techniques for cell isolation.Additionally, SC-β cells may also be enriched or isolated by methods ofserial subculture in growth conditions which promote the selectivesurvival or selective expansion of the SC-β cells. Such methods areknown by persons of ordinary skill in the art, and may include the useof agents such as, for example, insulin, members of the TGF-beta family,including Activin A, TGF-beta1, 2, and 3, bone morphogenic proteins(BMP-2, -3, -4, -5, -6, -7, -11, -12, and -13), fibroblast growthfactors-1 and -2, platelet-derived growth factor-AA, and —BB, plateletrich plasma, insulin-like growth factors (IGF-I, II) growthdifferentiation factor (GDF-5, -6, -7, -8, -10, -11, -15), vascularendothelial cell-derived growth factor (VEGF), Hepatocyte growth factor(HGF), pleiotrophin, endothelin, Epidermal growth factor (EGF),beta-cellulin, among others. Other pharmaceutical compounds can include,for example, nicotinamide, glucagon like peptide-I (GLP-1) and II, GLP-1and 2 mimetibody, Exendin-4, retinoic acid, parathyroid hormone.

Using the methods described herein, enriched, isolated and/or purifiedpopulations of SC-β cells can be produced in vitro from insulin-positiveendocrine cells or precursors thereof (which were differentiated frompluripotent stem cells by the methods described herein). In someembodiments, preferred enrichment, isolation and/or purification methodsrelate to the in vitro production of human SC-β cell from humaninsulin-positive endocrine cells or precursors thereof, which weredifferentiated from human pluripotent stem cells, or from human inducedpluripotent stem (iPS) cells. In such an embodiment, where SC-β cellsare differentiated from insulin-positive endocrine cells, which werepreviously derived from definitive endoderm cells, which were previouslyderived from iPS cells, the SC-β cell can be autologous to the subjectfrom whom the cells were obtained to generate the iPS cells.

Using the methods described herein, isolated cell populations of SC-βcells are enriched in SC-β cell content by at least about 2- to about1000-fold as compared to a population of cells before the chemicalinduction of the insulin-positive endocrine cell or precursorpopulation. In some embodiments, SC-β cells can be enriched by at leastabout 5- to about 500-fold as compared to a population before thechemical induction of an insulin-positive endocrine cell or precursorpopulation. In other embodiments, SC-β cells can be enriched from atleast about 10- to about 200-fold as compared to a population before thechemical induction of insulin-positive endocrine cell or precursorpopulation. In still other embodiments, SC-β cell can be enriched fromat least about 20- to about 100-fold as compared to a population beforethe chemical induction of insulin-positive endocrine cell or precursorpopulation. In yet other embodiments, SC-β cell can be enriched from atleast about 40- to about 80-fold as compared to a population before thechemical induction of insulin-positive endocrine cell or precursorpopulation. In certain embodiments, SC-β cell can be enriched from atleast about 2- to about 20-fold as compared to a population before thechemical induction of insulin-positive endocrine cell or precursorpopulation.

Compositions Comprising SC-β Cells

Some embodiments of the present invention relate to cell compositions,such as cell cultures or cell populations, comprising SC-β cells,wherein the SC-β cells have been derived from at least oneinsulin-positive endocrine cell or a precursor thereof. In someembodiments, the cell compositions comprise insulin-positive endocrinecells. In some embodiments, the cell compositions compriseNKX6-1-pancreatic progenitor cells. In some embodiments, the cellcompositions comprise Pdx1-pancreatic progenitor cells. In someembodiments, the cell compositions comprise primitive gut tube cells. Insome embodiments, the cell compositions comprise definitive endodermcells.

In accordance with certain embodiments, the chemically induced SC-βcells are mammalian cells, and in a preferred embodiment, such SC-βcells are human SC-β cells. In some embodiments, the insulin-positiveendocrine cells have been derived from definitive endoderm cells e.g.human definitive endoderm stem cells. In accordance with certainembodiments, the chemically induced Pdx1-positive pancreatic progenitorsare mammalian cells, and in a preferred embodiment, such Pdx1-positivepancreatic progenitors are human Pdx1-positive pancreatic progenitors.

Other embodiments of the present invention relate to compositions, suchas an isolated cell population or cell culture, comprising SC-β cellsproduced by the methods as disclosed herein. In some embodiments of thepresent invention relate to compositions, such as isolated cellpopulations or cell cultures, comprising chemically-induced SC-β cellsproduced by the methods as disclosed herein. In such embodiments, theSC-β cells comprise less than about 90%, less than about 85%, less thanabout 80%, less than about 75%, less than about 70%, less than about65%, less than about 60%, less than about 55%, less than about 50%, lessthan about 45%, less than about 40%, less than about 35%, less thanabout 30%, less than about 25%, less than about 20%, less than about15%, less than about 12%, less than about 10%, less than about 8%, lessthan about 6%, less than about 5%, less than about 4%, less than about3%, less than about 2% or less than about 1% of the total cells in theSC-β cells population. In some embodiments, the composition comprises apopulation of SC-β cells which make up more than about 90% of the totalcells in the cell population, for example about at least 95%, or atleast 96%, or at least 97%, or at least 98% or at least about 99%, orabout at least 100% of the total cells in the cell population are SC-βcells.

Certain other embodiments of the present invention relate tocompositions, such as an isolated cell population or cell cultures,comprise a combination of SC-β cells and insulin-positive endocrinecells or precursors thereof from which the SC-β cells were derived. Insome embodiments, the insulin-positive endocrine cells from which theSC-β cells are derived comprise less than about 25%, less than about20%, less than about 15%, less than about 10%, less than about 5%, lessthan about 4%, less than about 3%, less than about 2% or less than about1% of the total cells in the isolated cell population or culture.

Additional embodiments of the present invention relate to compositions,such as isolated cell populations or cell cultures, produced by theprocesses described herein and which comprise chemically induced SC-βcells as the majority cell type. In some embodiments, the methods andprocesses described herein produces an isolated cell culture and/or cellpopulations comprising at least about 99%, at least about 98%, at leastabout 97%, at least about 96%, at least about 95%, at least about 94%,at least about 93%, at least about 92%, at least about 91%, at leastabout 90%, at least about 89%, at least about 88%, at least about 87%,at least about 86%, at least about 85%, at least about 84%, at leastabout 83%, at least about 82%, at least about 81%, at least about 80%,at least about 79%, at least about 78%, at least about 77%, at leastabout 76%, at least about 75%, at least about 74%, at least about 73%,at least about 72%, at least about 71%, at least about 70%, at leastabout 69%, at least about 68%, at least about 67%, at least about 66%,at least about 65%, at least about 64%, at least about 63%, at leastabout 62%, at least about 61%, at least about 60%, at least about 59%,at least about 58%, at least about 57%, at least about 56%, at leastabout 55%, at least about 54%, at least about 53%, at least about 52%,at least about 51% or at least about 50% SC-β cells.

In another embodiment, isolated cell populations or compositions ofcells (or cell cultures) comprise human SC-β cells. In otherembodiments, the methods and processes as described herein can produceisolated cell populations comprising at least about 50%, at least about45%, at least about 40%, at least about 35%, at least about 30%, atleast about 25%, at least about 24%, at least about 23%, at least about22%, at least about 21%, at least about 20%, at least about 19%, atleast about 18%, at least about 17%, at least about 16%, at least about15%, at least about 14%, at least about 13%, at least about 12%, atleast about 11%, at least about 10%, at least about 9%, at least about8%, at least about 7%, at least about 6%, at least about 5%, at leastabout 4%, at least about 3%, at least about 2% or at least about 1% SC-βcells. In preferred embodiments, isolated cell populations can comprisehuman SC-β cells. In some embodiments, the percentage of SC-β cells inthe cell cultures or populations is calculated without regard to thefeeder cells remaining in the culture.

Still other embodiments of the present invention relate to compositions,such as isolated cell populations or cell cultures, comprising mixturesof SC-β cells and insulin-positive endocrine cells or precursors thereoffrom which they were differentiated from. For example, cell cultures orcell populations comprising at least about 5 SC-β cells for about every95 insulin-positive endocrine cells or precursors thereof can beproduced. In other embodiments, cell cultures or cell populationscomprising at least about 95 SC-β cells for about every 5insulin-positive endocrine cells or precursors thereof can be produced.Additionally, cell cultures or cell populations comprising other ratiosof SC-β cells to insulin-positive endocrine cells or precursors thereofare contemplated. For example, compositions comprising at least about 1SC-β cell for about every 1,000,000, or at least 100,000 cells, or aleast 10,000 cells, or at least 1000 cells or 500, or at least 250 or atleast 100 or at least 10 insulin-positive endocrine cells or precursorsthereof can be produced.

Further embodiments of the present invention relate to compositions,such as cell cultures or cell populations, comprising human cells,including human SC-β cell which displays at least one characteristic ofan endogenous β cell.

In preferred embodiments of the present invention, cell cultures and/orcell populations of SC-β cells comprise human SC-β cells that arenon-recombinant cells. In such embodiments, the cell cultures and/orcell populations are devoid of or substantially free of recombinanthuman SC-β cells.

β Cell Maturation Factors

Aspects of the disclosure involve contacting insulin-positive endocrinecells or precursors thereof with β cell maturation factors, for example,to induce the maturation of the insulin-positive endocrine cells ordifferentiation of the precursors thereof into SC-β cells (e.g., maturepancreatic β cells). The term “β cell maturation factor” refers to anagent that promotes or contributes to conversion of at least oneinsulin-positive endocrine cell or a precursor thereof to a SC-β cell.In some embodiments, the β cell maturation factor induces thedifferentiation of pluripotent cells (e.g., iPSCs or hESCs) intodefinitive endoderm cells, e.g., in accordance with a method describedherein. In some embodiments, the β cell maturation factor induces thedifferentiation of definitive endoderm cells into primitive gut tubecells, e.g., in accordance with a method described herein. In someembodiments, the β cell maturation factor induces the differentiation ofprimitive gut tube cells into Pdx1-positive pancreatic progenitor cells,e.g., in accordance with a method described herein. In some embodiments,the β cell maturation factor induces the differentiation ofPdx1-positive pancreatic progenitor cells into NKX6-1-positivepancreatic progenitor cells, e.g., in accordance with a method describedherein. In some embodiments, the β cell maturation factor induces thedifferentiation of NKX6-1-positive pancreatic progenitor cells intoinsulin-positive endocrine cells, e.g., in accordance with a methoddescribed herein. In some embodiments, the β cell maturation factorinduces the maturation of insulin-positive endocrine cells into SC-βcells, e.g., in accordance with a method described herein.

Generally, at least one β cell maturation factor described herein can beused alone, or in combination with other β cell maturation factors, togenerate SC-β cells according to the methods as disclosed herein. Insome embodiments, at least two, at least three, at least four, at leastfive, at least six, at least seven, at least eight, at least nine, or atleast ten β cell maturation factors described herein are used in themethods of generating SC-β cells.

Transforming Growth Factor-β (TGF-β) Superfamily

Aspects of the disclosure relate to the use of growth factors from thetransforming growth factor-β (TGF-β) superfamily as β cell maturationfactors. The “TGF-β superfamily” means proteins having structural andfunctional characteristics of known TGFβ family members. The TGFβ familyof proteins is well characterized, both from structural and functionalaspects. It includes the TGFβ series of proteins, the Inhibins(including Inhibin A and Inhibin B), the Activins (including Activin A,Activin B, and Activin AB), MIS (Müllerian inhibiting substance), BMP(bone morphogenetic proteins), dpp (decapentaplegic), Vg-1, MNSF(monoclonal nonspecific suppressor factor), and others. Activity of thisfamily of proteins is based on specific binding to certain receptors onvarious cell types. Members of this family share regions of sequenceidentity, particularly at the C-terminus, that correlate to theirfunction. The TGFβ family includes more than one hundred distinctproteins, all sharing at least one region of amino acid sequenceidentity. Members of the family include, but are not limited to, thefollowing proteins, as identified by their GenBank accession numbers:P07995, P18331, P08476, Q04998, P03970, P43032, P55102, P27092, P42917,P09529, P27093, P04088, Q04999, P17491, P55104, Q9WUK5, P55103, O88959,O08717, P58166, O61643, P35621, P09534, P48970, Q9NR23, P25703, P30884,P12643, P49001, P21274, O46564, O19006, P22004, P20722, Q04906, Q07104,P30886, P18075, P23359, P22003, P34821, P49003, Q90751, P21275, Q06826,P30885, P34820, Q29607, P12644, Q90752, O46576, P27539, P48969, Q26974,P07713, P91706, P91699, P27091, O42222, Q24735, P20863, O18828, P55106,Q9PTQ2, O14793, O08689, O42221, O18830, O18831, O18836, O35312, O42220,P43026, P43027, P43029, O95390, Q9R229, O93449, Q9Z1W4, Q9BDW8, P43028,Q7Z4P5, P50414, P17246, P54831, P04202, P01137, P09533, P18341, O19011,Q9Z1Y6, P07200, Q9Z217, O95393, P55105, P30371, Q9MZE2, Q07258, Q96S42,P97737, AAA97415.1, NP-776788.1, NP-058824.1, EAL24001.1, 1S4Y,NP-001009856.1, NP-032406.1, NP-999193.1, XP 519063.1, AAG17260.1,CAA40806.1, NP-001009458.1, AAQ55808.1, AAK40341.1, AAP33019.1,AAK21265.1, AAC59738.1, CA46003.1, B40905, AAQ55811.1, AAK40342.1,XP-540364.1, P55102, AAQ55810.1, NP-990727.1, CAA51163.1, AAD50448.1,JC4862, PN0504, BAB17600.1, AAH56742.1, BAB17596.1, CAG06183.1,CAG05339.1, BAB17601.1, CAB43091.1, A36192, AAA49162.1, AAT42200.1,NP-789822.1, AAA59451.1, AAA59169.1, XP 541000.1, NP-990537.1,NP-002184.1, AAC14187.1, AAP83319.1, AAA59170.1, BAB16973.1, AAM66766.1,WFPGBB, 1201278C, AAH30029.1, CAA49326.1, XP-344131.1, AAH48845.1,XP-148966.3, I48235, B41398, AAH77857.1, AAB26863.1, 1706327A,BAA83804.1, NP-571143.1, CAG00858.1, BAB17599.1, BAB17602.1, AAB61468.1,PN0505, PN0506, CAB43092.1, BAB17598.1, BAA22570.1, BAB16972.1,BAC81672.1, BAA12694.1, BAA08494.1, B36192, C36192, BAB16971.1,NP-034695.1, AAA49160.1, CAA62347.1, AAA49161.1, AAD30132.1, CAA58290.1,NP-005529.1, XP-522443.1, AAM27448.1, XP 538247.1, AAD30133.1,AAC36741.1, AAH10404.1, NP-032408.1, AAN03682.1, XP-509161.1,AAC32311.1, NP-651942.2, AAL51005.1, AAC39083.1, AAH85547.1,NP-571023.1, CAF94113.1, EAL29247.1, AAW30007.1, AAH90232.1, A29619,NP-001007905.1, AAH73508.1, AAD02201.1, NP 999793.1, NP-990542.1,AAF19841.1, AAC97488.1, AAC60038.1, NP 989197.1, NP-571434.1,EAL41229.1, AAT07302.1, CA119472.1, NP-031582.1, AAA40548.1,XP-535880.1, NP-037239.1, AAT72007.1, XP-418956.1, CAA41634.1,BAC30864.1, CAA38850.1, CAB81657.2, CAA45018.1, CAA45019.1, BAC28247.1,NP-031581.1, NP-990479.1, NP-999820.1, AAB27335.1, S45355, CAB82007.1,XP-534351.1, NP-058874.1, NP-031579.1, 1REW, AAB96785.1, AAB46367.1,CAA05033.1, BAA89012.1, 1ES7, AAP20870.1, BAC24087.1, AAG09784.1,BAC06352.1, AAQ89234.1, AAM27000.1, AAH30959.1, CAG01491.1, NP-571435.1,1REU, AAC60286.1, BAA24406.1, A36193, AAH55959.1, AAH54647.1,AAH90689.1, CAG09422.1, BAD16743.1, NP-032134.1, XP-532179.1,AAB24876.1, AAH57702.1, AAA82616.1, CAA40222.1, CAB90273.2, XP-342592.1,XP-534896.1, XP 534462.1, 1LXI, XP-417496.1, AAF34179.1, AAL73188.1,CAF96266.1, AAB34226.1, AAB33846.1, AAT12415.1, AA33819.1, AAT72008.1,AAD38402.1, BAB68396.1, CAA45021.1, AAB27337.1, AAP69917.1, AAT12416.1,NP-571396.1, CAA53513.1, AA033820.1, AAA48568.1, BAC02605.1, BAC02604.1,BAC02603.1, BAC02602.1, BAC02601.1, BAC02599.1, BAC02598.1, BAC02597.1,BAC02595.1, BAC02593.1, BAC02592.1, BAC02590.1, AAD28039.1, AAP74560.1,AAB94786.1, NP-001483.2, XP-528195.1, NP 571417.1, NP-001001557.1,AAH43222.1, AAM33143.1, CAG10381.1, BAA31132.1, EAL39680.1, EAA12482.2,P34820, AAP88972.1, AAP74559.1, CAI16418.1, AAD30538.1, XP-345502.1,NP-038554.1, CAG04089.1, CAD60936.2, NP-031584.1, B55452, AAC60285.1,BAA06410.1, AAH52846.1, NP-031580.1, NP-036959.1, CAA45836.1,CAA45020.1, Q29607, AAB27336.1, XP-547817.1, AAT12414.1, AAM54049.1,AAH78901.1, AA025745.1, NP-570912.1, XP-392194.1, AAD20829.1,AAC97113.1, AAC61694.1, AAH60340.1, AAR97906.1, BAA32227.1, BAB68395.1,BAC02895.1, AAW51451.1, AAF82188.1, XP-544189.1, NP-990568.1,BAC80211.1, AAW82620.1, AAF99597.1, NP-571062.1, CAC44179.1, AAB97467.1,AAT99303.1, AAD28038.1, AAH52168.1, NP-001004122.1, CAA72733.1,NP-032133.2, XP 394252.1, XP-224733.2, JH0801, AAP97721.1, NP-989669.1,S43296, P43029, A55452, AAH32495.1, XP-542974.1, NP-032135.1,AAK30842.1, AAK27794.1, BAC30847.1, EAA12064.2, AAP97720.1, XP-525704.1,AAT07301.1, BAD07014.1, CAF94356.1, AAR27581.1, AAG13400.1, AAC60127.1,CAF92055.1, XP-540103.1, AA020895.1, CAF97447.1, AAS01764.1, BAD08319.1,CAA10268.1, NP-998140.1, AAR03824.1, AAS48405.1, AAS48403.1, AAK53545.1,AAK84666.1, XP-395420.1, AAK56941.1, AAC47555.1, AAR88255.1, EAL33036.1,AAW47740.1, AAW29442.1, NP 722813.1, AAR08901.1, AA015420.2, CAC59700.1,AAL26886.1, AAK71708.1, AAK71707.1, CAC51427.2, AAK67984.1, AAK67983.1,AAK28706.1, P07713, P91706, P91699, CAG02450.1, AAC47552.1, NP-005802.1,XP-343149.1, AW34055.1, XP-538221.1, AAR27580.1, XP-125935.3,AAF21633.1, AAF21630.1, AAD05267.1, Q9Z1W4, NP-031585.2, NP-571094.1,CAD43439.1, CAF99217.1, CAB63584.1, NP-722840.1, CAE46407.1,XP-417667.1, BAC53989.1, BAB19659.1, AAM46922.1, AAA81169.1, AAK28707.1,AAL05943.1, AAB17573.1, CAH25443.1, CAG10269.1, BAD16731.1, EAA00276.2,AAT07320.1, AAT07300.1, AAN15037.1, CAH25442.1, AAK08152.2, 2009388A,AAR12161.1, CAG01961.1, CAB63656.1, CAD67714.1, CAF94162.1, NP-477340.1,EAL24792.1, NP-001009428.1, AAB86686.1, AAT40572.1, AAT40571.1,AAT40569.1, NP-033886.1, AAB49985.1, AAG39266.1, Q26974, AAC77461.1,AAC47262.1, BAC05509.1, NP-055297.1, XP-546146.1, XP-525772.1,NP-060525.2, AAH33585.1, AAH69080.1, CAG12751.1, AAH74757.2,NP-034964.1, NP-038639.1, 042221, AAF02773.1, NP-062024.1, AAR18244.1,AAR14343.1, XP-228285.2, AAT40573.1, AAT94456.1, AAL35278.1, AAL35277.1,AAL17640.1, AAC08035.1, AAB86692.1, CAB40844.1, BAC38637.1, BAB16046.1,AAN63522.1, NP 571041.1, AAB04986.2, AAC26791.1, AAB95254.1, BAA11835.1,AAR18246.1, XP-538528.1, BAA31853.1, AAK18000.1, XP-420540.1,AAL35276.1, AAQ98602.1, CAE71944.1, AAW50585.1, AAV63982.1, AAW29941.1,AAN87890.1, AAT40568.1, CAD57730.1, AAB81508.1, AAS00534.1, AAC59736.1,BAB79498.1, AAA97392.1, AAP85526.1, NP-999600.2, NP 878293.1,BAC82629.1, CAC60268.1, CAG04919.1, AAN10123.1, CAA07707.1 AAK20912.1,AAR88254.1, CAC34629.1, AAL35275.1, AAD46997.1, AAN03842.1, NP-571951.2,CAC50881.1, AAL99367.1, AAL49502.1, AAB71839.1, AAB65415.1, NP-624359.1,NP-990153.1, AAF78069.1, AAK49790.1, NP-919367.2, NP-001192.1,XP-544948.1, AAQ18013.1, AAV38739.1, NP-851298.1, CAA67685.1,AAT67171.1, AAT37502.1, AAD27804.1, AAN76665.1, BAC11909.1, XP-421648.1,CAB63704.1, NP-037306.1, A55706, AAF02780.1, CAG09623.1, NP-067589.1,NP-035707.1, AAV30547.1, AAP49817.1, BAC77407.1, AAL87199.1, CAG07172.1,B36193, CAA33024.1, NP-001009400.1, AAP36538.1, XP-512687.1,XP-510080.1, AAH05513.1, 1KTZ, AAH14690.1, AAA31526.1.

It is contemplated that any growth factor from the TGF-β superfamilythat is capable, either alone or in combination with one or more other βcell maturation factors, of inducing the differentiation of at least oneinsulin-producing, endocrine cell or precursor thereof into a SC-β cellcan be used in the methods, compositions, and kits described herein.

The growth factor from the TGF-β can be naturally obtained orrecombinant. In some embodiments, the growth factor from the TGF-βsuperfamily comprises Activin A. The term “Activin A” includes fragmentsand derivatives of Activin A. The sequence of an exemplary Activin A isdisclosed as SEQ ID NO: 1 in U.S. Pub. No. 2009/0155218 (the '218publication’). Other non-limiting examples of Activin A are provided inSEQ ID NO: 2-16 of the '218 publication, and non-limiting examples ofnucleic acids encoding Activin A are provided in SEQ ID NO:33-34 of the'218 publication. In some embodiments, the growth factor from the TGF-βsuperfamily comprises a polypeptide having an amino acid sequence atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, or at least 99%, or greateridentical to SEQ ID NO: 1 of the '218 publication.

In some embodiments, the growth factor from the TGF-β superfamilycomprises growth differentiation factor 8 (GDF8). The term “GDF8”includes fragments and derivatives of GDF8. The sequences of GDF8polypeptides are available to the skilled artisan. In some embodiments,the growth factor from the TGF-β superfamily comprises a polypeptidehaving an amino acid sequence at least 30%, at least 40%, at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, orat least 99%, or greater identical to the human GDF8 polypeptidesequence (GenBank Accession EAX10880).

In some embodiments, the growth factor from the TGF-β superfamilycomprises a growth factor that is closely related to GDF8, e.g., growthdifferentiation factor 11 (GDF11). The polypeptide sequences of GDF11are available to the skilled artisan. In some embodiments, the growthfactor from the TGF-β superfamily comprises a polypeptide having anamino acid sequence at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least99%, or greater identical to the human GDF11 polypeptide sequence(GenBank Accession AAF21630).

In certain embodiments, the methods, compositions, and kits disclosedherein exclude at least one growth factor from the TGF-β superfamily.

In some embodiments, the at least one growth factor from the TGF-βsuperfamily can be replaced with an agent mimics the at least one growthfactor from the TGF-β superfamily. Exemplary agents that mimic the atleast one growth factor from the TGF-β superfamily, include, withoutlimitation, IDE1 and IDE2

TGF-β Signaling Pathway Inhibitors

Aspects of the disclosure relate to the use of TGF-β signaling pathwayinhibitors as β cell maturation factors. It is contemplated that anyTGF-β signaling pathway inhibitor that is capable, either alone or incombination with one or more other β cell maturation factors, ofinducing the differentiation of at least one insulin-producing,endocrine cell or precursor thereof into a SC-β cell can be used in themethods, compositions, and kits described herein. In certainembodiments, the methods, compositions, and kits disclosed hereinexclude a TGF-β signaling pathway inhibitor.

In some embodiments, the TGF-β signaling pathway comprises TGF-βreceptor type I kinase (TGF-β RI) signaling. In some embodiments, theTGF-β signaling pathway inhibitor comprises ALK5 inhibitor II (CAS446859-33-2, an ATP-competitive inhibitor of TGF-β RI kinase, also knownas RepSox, IUPAC Name:2-[5-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl]-1,5-naphthyridine. In someembodiments, the TGF-β signaling pathway inhibitor is an analog orderivative of ALK5 inhibitor II.

In some embodiments, the analog or derivative of ALK5 inhibitor II is acompound of Formula I as described in U.S. Patent Publication No.2012/0021519, incorporated by reference herein in its entirety.

In some embodiments, the TGF-β signaling pathway inhibitor is a TGF-βreceptor inhibitor described in U.S. Patent Publication No.2010/0267731. In some embodiments, the TGF-β signaling pathway inhibitorcomprises an ALK5 inhibitor described in U.S. Patent Publication Nos.2009/0186076 and 2007/0142376.

In some embodiments, the TGF-β signaling pathway inhibitor is A 83-01.In some embodiments, the TGF-β signaling pathway inhibitor is not A83-01. In some embodiments, the compositions and methods describedherein exclude A 83-01.

In some embodiments, the TGF-β signaling pathway inhibitor is SB 431542.In some embodiments, the TGF-β signaling pathway inhibitor is not SB431542. In some embodiments, the compositions and methods describedherein exclude SB 431542.

In some embodiments, the TGF-β signaling pathway inhibitor is D 4476. Insome embodiments, the TGF-β signaling pathway inhibitor is not D 4476.In some embodiments, the compositions and methods described hereinexclude D 4476.

In some embodiments, the TGF-β signaling pathway inhibitor is GW 788388.In some embodiments, the TGF-β signaling pathway inhibitor is not GW788388. In some embodiments, the compositions and methods describedherein exclude GW 788388.

In some embodiments, the TGF-β signaling pathway inhibitor is LY 364947.In some embodiments, the TGF-β signaling pathway inhibitor is not LY364947. In some embodiments, the compositions and methods describedherein exclude LY 364947.

In some embodiments, the TGF-β signaling pathway inhibitor is LY 580276.In some embodiments, the TGF-β signaling pathway inhibitor is not LY580276. In some embodiments, the compositions and methods describedherein exclude LY 580276.

In some embodiments, the TGF-β signaling pathway inhibitor is SB 525334.In some embodiments, the TGF-β signaling pathway inhibitor is not SB525334. In some embodiments, the compositions and methods describedherein exclude SB 525334.

In some embodiments, the TGF-β signaling pathway inhibitor is SB 505124.In some embodiments, the TGF-β signaling pathway inhibitor is not SB505124. In some embodiments, the compositions and methods describedherein exclude SB 505124.

In some embodiments, the TGF-β signaling pathway inhibitor is SD 208. Insome embodiments, the TGF-β signaling pathway inhibitor is not SD 208.In some embodiments, the compositions and methods described hereinexclude SD 208.

In some embodiments, the TGF-β signaling pathway inhibitor is GW 6604.In some embodiments, the TGF-β signaling pathway inhibitor is not GW6604. In some embodiments, the compositions and methods described hereinexclude GW 6604.

In some embodiments, the TGF-β signaling pathway inhibitor is GW 788388.In some embodiments, the TGF-β signaling pathway inhibitor is not GW788388. In some embodiments, the compositions and methods describedherein exclude GW 788388.

From the collection of compounds described above, the following can beobtained from various sources: LY-364947, SB-525334, SD-208, andSB-505124 available from Sigma, P.O. Box 14508, St. Louis, Mo.,63178-9916; 616452 and 616453 available from Calbiochem (EMD Chemicals,Inc.), 480 S. Democrat Road, Gibbstown, N.J., 08027; GW788388 and GW6604available from GlaxoSmithKline, 980 Great West Road, Brentford,Middlesex, TW8 9GS, United Kingdom; LY580276 available from LillyResearch, Indianapolis, Ind. 46285; and SM16 available from Biogen Idec,P.O. Box 14627, 5000 Davis Drive, Research Triangle Park, N.C.,27709-4627.

WNT Signaling Pathway

Aspects of the disclosure relate to the use of activators of the WNTsignaling pathway as β cell maturation factors. It is contemplated thatany WNT signaling pathway activator that is capable, either alone or incombination with one or more other β cell maturation factors, ofinducing the differentiation of at least one insulin-producing,endocrine cell or precursor thereof into a SC-β cell can be used in themethods, compositions, and kits described herein.

In some embodiments, the WNT signaling pathway activator comprisesCHIR99021. In some embodiments, the WNT signaling pathway activatorcomprises a derivative of CHIR99021, e.g., a salt of CHIR99021, e.g.,trihydrochloride, a hydrochloride salt of CHIR99021. In someembodiments, the WNT signaling pathway activator comprises Wnt3arecombinant protein. In some embodiments, the WNT signaling pathwayactivator comprises a glycogen synthase kinase 3 (GSK3) inhibitor.Exemplary GSK3 inhibitors include, without limitation, 3F8, A 1070722,AR-A 014418, BIO, BIO-acetoxime, FRATide, 10Z-Hymenialdisine,Indirubin-3′oxime, kenpaullone, L803, L803-mts, lithium carbonate, NSC693868, SB 216763, SB 415286, TC-G 24, TCS 2002, TCS 21311, TWS 119, andanalogs or derivatives of any of these. In certain embodiments, themethods, compositions, and kits disclosed herein exclude a WNT signalingpathway activator.

Fibroblast Growth Factor (FGF) Family

Aspects of the disclosure relate to the use of growth factors from theFGF family as β cell maturation factors. It is contemplated that anygrowth factor from the FGF family that is capable, either alone or incombination with one or more other β cell maturation factors, ofinducing the differentiation of at least one insulin-producing,endocrine cell or precursor thereof into a SC-β cell can be used in themethods, compositions, and kits described herein. In certainembodiments, the methods, compositions, and kits disclosed hereinexclude a growth factor from the FGF family.

In some embodiments, the at least one growth factor from the FGF familycomprises keratinocyte growth factor (KGF). The polypeptide sequences ofKGF are available to the skilled artisan. In some embodiments, thegrowth factor from the FGF family comprises a polypeptide having anamino acid sequence at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least99%, or greater identical to the human KGF polypeptide sequence (GenBankAccession AAB21431).

In some embodiments, the at least one growth factor from the FGF familycomprises FGF2. The polypeptide sequences of FGF2 are available to theskilled artisan. In some embodiments, the growth factor from the FGFfamily comprises a polypeptide having an amino acid sequence at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, or at least 99%, or greater identicalto the human FGF2 polypeptide sequence (GenBank Accession NP_001997).

In some embodiments, the at least one growth factor from the FGF familycomprises FGF8B. The polypeptide sequences of FGF8B are available to theskilled artisan. In some embodiments, the growth factor from the FGFfamily comprises a polypeptide having an amino acid sequence at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, or at least 99%, or greater identicalto the human FGF8B polypeptide sequence (GenBank Accession AAB40954).

In some embodiments, the at least one growth factor from the FGF familycomprises FGF10. The polypeptide sequences of FGF10 are available to theskilled artisan. In some embodiments, the growth factor from the FGFfamily comprises a polypeptide having an amino acid sequence at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, or at least 99%, or greater identicalto the human FGF10 polypeptide sequence (GenBank Accession CAG46489).

In some embodiments, the at least one growth factor from the FGF familycomprises FGF21. The polypeptide sequences of FGF21 are available to theskilled artisan. In some embodiments, the growth factor from the FGFfamily comprises a polypeptide having an amino acid sequence at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, or at least 99%, or greater identicalto the human FGF21 polypeptide sequence (GenBank Accession AAQ89444.1).

Bone Morphogenic Protein (BMP) Signaling Pathway Inhibitors

Aspects of the disclosure relate to the use of BMP signaling pathwayinhibitors as β cell maturation factors. The BMP signaling family is adiverse subset of the TGF-β superfamily (Sebald et al. Biol. Chem.385:697-710, 2004). Over twenty known BMP ligands are recognized bythree distinct type II (BMPRII, ActRIIa, and ActRIIb) and at least threetype I (ALK2, ALK3, and ALK6) receptors. Dimeric ligands facilitateassembly of receptor heteromers, allowing the constitutively-active typeII receptor serine/threonine kinases to phosphorylate type I receptorserine/threonine kinases. Activated type I receptors phosphorylateBMP-responsive (BR-) SMAD effectors (SMADs 1, 5, and 8) to facilitatenuclear translocation in complex with SMAD4, a co-SMAD that alsofacilitates TGF signaling. In addition, BMP signals can activateintracellular effectors such as MAPK p38 in a SMAD-independent manner(Nohe et al. Cell Signal 16:291-299, 2004). Soluble BMP antagonists suchas noggin, chordin, gremlin, and follistatin limit BMP signaling byligand sequestration.

It is contemplated that any BMP signaling pathway inhibitor that iscapable, either alone or in combination with one or more other β cellmaturation factors, of inducing the differentiation of at least oneinsulin-producing, endocrine cell or precursor thereof into a SC-β cellcan be used in the methods, compositions, and kits described herein. Incertain embodiments of any aspect described herein, the methods,compositions, and kits disclosed herein exclude a BMP signaling pathwayinhibitor.

In some embodiments, the BMP signaling pathway inhibitor comprises LDN193189 (also known as LDN193189, 1062368-24-4, LDN-193189, DM 3189,DM-3189, IUPAC Name:4-[6-(4-piperazin-1-ylphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinolone).

In some embodiments, the BMP signaling pathway inhibitor comprise ananalog or derivative of LDN 193189, e.g., a salt, hydrate, solvent,ester, or prodrug of LDN 193189. In some embodiments, a derivative(e.g., salt) of LDN 193189 comprises LDN193189 hydrochloride.

In some embodiments, the BMP signaling pathway inhibitor comprises acompound of Formula I from U.S. Patent Publication No. 2011/0053930.

Sonic Hedgehog (SHH) Signaling Pathway

Aspects of the disclosure relate to the use of SHH signaling pathwayinhibitors as β cell maturation factors. It is contemplated that any SHHsignaling pathway inhibitor that is capable, either alone or incombination with one or more other β cell maturation factors, ofinducing the differentiation of at least one insulin-producing,endocrine cell or precursor thereof into a SC-β cell can be used in themethods, compositions, and kits described herein.

In some embodiments, the SHH signaling pathway inhibitor comprisesSant1. In some embodiments, the SHH signaling pathway inhibitorcomprises SANT2. In some embodiments, the SHH signaling pathwayinhibitor comprises SANT3. In some embodiments, the SHH signalingpathway inhibitor comprises SANT4. In some embodiments, the SHHsignaling pathway inhibitor comprises Cur61414. In some embodiments, theSHH signaling pathway inhibitor comprises forskolin. In someembodiments, the SHH signaling pathway inhibitor comprises tomatidine.In some embodiments, the SHH signaling pathway inhibitor comprisesAY9944. In some embodiments, the SHH signaling pathway inhibitorcomprises triparanol. In some embodiments, the SHH signaling pathwayinhibitor comprises compound A or compound B (as disclosed in U.S. Pub.No. 2004/0060568). In some embodiments, the SHH signaling pathwayinhibitor comprises a steroidal alkaloid that antagonizes hedgehogsignaling (e.g., cyclopamine or a derivative thereof) as disclosed inU.S. Pub. No. 2006/0276391. In certain embodiments, the methods,compositions, and kits disclosed herein exclude a SHH signaling pathwayinhibitor.

Retinoic Acid Signaling Pathway

Aspects of the disclosure relate to the use of modulators of retinoicacid signaling as β cell maturation factors. It is contemplated that anymodulator of retinoic acid signaling that is capable, either alone or incombination with one or more other β cell maturation factors, ofinducing the differentiation of at least one insulin-producing,endocrine cell or precursor thereof into a SC-β cell can be used in themethods, compositions, and kits described herein.

In some embodiments, the modulator of retinoic acid signaling comprisesan activator of retinoic acid signaling. In some embodiments, the RAsignaling pathway activator comprises retinoic acid. In someembodiments, the RA signaling pathway activator comprises a retinoicacid receptor agonist. Exemplary retinoic acid receptor agonistsinclude, without limitation, CD 1530, AM 580, TTNPB, CD 437, Ch 55, BMS961, AC 261066, AC 55649, AM 80, BMS 753, tazarotene, adapalene, and CD2314.

In some embodiments, the modulator of retinoic acid signaling comprisesan inhibitor of retinoic acid signaling. In some embodiments, theretinoic acid signaling pathway inhibitor comprises DEAB (IUPAC Name:2-[2-(diethylamino)ethoxy]-3-prop-2-enylbenzaldehyde). In someembodiments, the retinoic acid signaling pathway inhibitor comprises ananalog or derivative of DEAB.

In some embodiments, the retinoic acid signaling pathway inhibitorcomprises a retinoic acid receptor antagonist. In some embodiments, theretinoic acid receptor antagonist comprises(E)-4-[2-(5,6-dihydro-5,5-dimethyl-8-phenyl-2-naphthalenyl)ethenyl]benzoicacid,(E)-4-[[(5,6-dihydro-5,5-dimethyl-8-phenylethynyl)-2-naphthalenyl]ethenyl]benzoicacid,(E)-4-[2-[5,6-dihydro-5,5-dimethyl-8-(2-naphthalenyl)-2-naphthalenyl]ethenyl]-benzoicacid, and(E)-4-[2-[5,6-dihydro-5,5-dimethyl-8-(4-methoxyphenyl)-2-naphthalenyl]ethenyl]benzoicacid. In some embodiments, the retinoic acid receptor antagonistcomprises BMS 195614 (CAS #253310-42-8), ER 50891 (CAS #187400-85-7),BMS 493 (CAS #170355-78-9), CD 2665 (CAS #170355-78-9), LE 135 (CAS#155877-83-1), BMS 453 (CAS #166977-43-1), or MM 11253 (CAS#345952-44-5).

In certain embodiments, the methods, compositions, and kits disclosedherein exclude a modulator of retinoic acid signaling. In certainembodiments, the methods, compositions, and kits disclosed hereinexclude a retinoic acid signaling pathway activator. In certainembodiments, the methods, compositions, and kits disclosed hereinexclude a retinoic acid signaling pathway inhibitor.

Protein Kinase C

Aspects of the disclosure relate to the use of protein kinase Cactivators as β cell maturation factors. Protein kinase C is one of thelargest families of protein kinase enzymes and is composed of a varietyof isoforms. Conventional isoforms include a, βI, βII, γ; novel isoformsinclude δ, ε, η, Θ; and atypical isoforms include ξ, and ι/λ. PKCenzymes are primarily cytosolic but translocate to the membrane whenactivated. In the cytoplasm, PKC is phosphorylated by other kinases orautophosphorylates. In order to be activated, some PKC isoforms (e.g.,PKC-ε) require a molecule to bind to the diacylglycerol (“DAG”) bindingsite or the phosphatidylserine (“PS”) binding site. Others are able tobe activated without any secondary binding messengers at all. PKCactivators that bind to the DAG site include, but are not limited to,bryostatin, picologues, phorbol esters, aplysiatoxin, and gnidimacrin.PKC activators that bind to the PS site include, but are not limited to,polyunsaturated fatty acids and their derivatives. It is contemplatedthat any protein kinase C activator that is capable, either alone or incombination with one or more other β cell maturation factors, ofinducing the differentiation of at least one insulin-producing,endocrine cell or precursor thereof into a SC-β cell can be used in themethods, compositions, and kits described herein.

In some embodiments, the PKC activator comprises PdbU. In someembodiments, the PKC activator comprises TPB. In some embodiments, thePKC activator comprises cyclopropanated polyunsaturated fatty acids,cyclopropanated monounsaturated fatty acids, cyclopropanatedpolyunsaturated fatty alcohols, cyclopropanated monounsaturated fattyalcohols, cyclopropanated polyunsaturated fatty acid esters,cyclopropanated monounsaturated fatty acid esters, cyclopropanatedpolyunsaturated fatty acid sulfates, cyclopropanated monounsaturatedfatty acid sulfates, cyclopropanated polyunsaturated fatty acidphosphates, cyclopropanated monounsaturated fatty acid phosphates,macrocyclic lactones, DAG derivatives, isoprenoids, octylindolactam V,gnidimacrin, iripallidal, ingenol, napthalenesulfonamides,diacylglycerol kinase inhibitors, fibroblast growth factor 18 (FGF-18),insulin growth factor, hormones, and growth factor activators, asdescribed in WIPO Pub. No. WO/2013/071282. In some embodiments, thebryostain comprises bryostatin-1, bryostatin-2, bryostatin-3,bryostatin-4, bryostatin-5, bryostatin-6, bryostatin-7, bryostatin-8,bryostatin-9, bryostatin-10, bryostatin-11, bryostatin-12,bryostatin-13, bryostatin-14, bryostatin-15, bryostatin-16,bryostatin-17, or bryostatin-18. In certain embodiments, the methods,compositions, and kits disclosed herein exclude a protein kinase Cactivator.

γ-Secretase Inhibitors

Aspects of the disclosure relate to the use of γ-secretase inhibitors asβ cell maturation factors. It is contemplated that any γ-secretaseinhibitor that is capable, either alone or in combination with one ormore other β cell maturation factors, of inducing the differentiation ofat least one insulin-producing, endocrine cell or precursor thereof intoa SC-β cell can be used in the methods, compositions, and kits describedherein. Numerous γ-secretase inhibitors are known. In some embodiments,the γ-secretase inhibitor comprises XXI. In some embodiments, theγ-secretase inhibitor comprises DAPT. Additional exemplary γ-secretaseinhibitors include, without limitation, the γ-secretase inhibitorsdescribed in U.S. Pat. Nos. 7,049,296, 8,481,499, 8,501,813, and WIPOPub. No. WO/2013/052700. In certain embodiments, the methods,compositions, and kits disclosed herein exclude a 7-secretase inhibitor.

Thyroid Hormone Signaling Pathway Activators

Aspects of the disclosure relate to the use of thyroid hormone signalingpathway activators as β cell maturation factors. It is contemplated thatany thyroid hormone signaling pathway activator that is capable, eitheralone or in combination with one or more other β cell maturationfactors, of inducing the differentiation of at least oneinsulin-positive endocrine cell or precursor thereof into a SC-β cellcan be used in the methods, compositions, and kits described herein. Incertain embodiments of any aspect described herein, the methods,compositions, and kits disclosed herein exclude a thyroid hormonesignaling pathway activator. In certain embodiments of any aspectdescribed herein, the methods, compositions, and kits disclosed hereinexclude T3 or an analog of T3 described herein.

In some embodiments, the thyroid hormone signaling pathway activatorcomprises triiodothyronine (T3). In some embodiments, the thyroidhormone signaling pathway activator comprises an analog or derivative ofT3. Exemplary analogs of T3 include, but are not limited to, selectiveand non-selective thyromimetics, TRO selective agonist-GC-1,GC-24,4-Hydroxy-PCB 106, MB07811, MB07344,3,5-diiodothyropropionic acid(DITPA); the selective TR-β agonist GC-1; 3-Iodothyronamine (T(1)AM) and3,3′,5-triiodothyroacetic acid (Triac) (bioactive metabolites of thehormone thyroxine (T(4)); KB-2115 and KB-141; thyronamines; SKF L-94901;DIBIT; 3′-AC-T2; tetraiodothyroacetic acid (Tetrac) andtriiodothyroacetic acid (Triac) (via oxidative deamination anddecarboxylation of thyroxine [T4] and triiodothyronine [T3] alaninechain), 3,3′,5′-triiodothyronine (rT3) (via T4 and T3 deiodination),3,3′-diiodothyronine (3,3′-T2) and 3,5-diiodothyronine (T2) (via T4, T3,and rT3 deiodination), and 3-iodothyronamine (T1AM) and thyronamine(T0AM) (via T4 and T3 deiodination and amino acid decarboxylation), aswell as for TH structural analogs, such as 3,5,3′-triiodothyropropionicacid (Triprop), 3,5-dibromo-3-pyridazinone-1-thyronine (L-940901),N-[3,5-dimethyl-4-(4′-hydroxy-3′-isopropylphenoxy)-phenyl]-oxamic acid(CGS 23425),3,5-dimethyl-4-[(4′-hydroxy-3′-isopropylbenzyl)-phenoxy]acetic acid(GC-1), 3,5-dichloro-4-[(4-hydroxy-3-isopropylphenoxy)phenyl]acetic acid(KB-141), and 3,5-diiodothyropropionic acid (DITPA).

In some embodiments, the thyroid hormone signaling pathway activatorcomprises a prodrug or prohormone of T3, such as T4 thyroid hormone(e.g., thyroxine or L-3,5,3′,5′-tetraiodothyronine).

In some embodiments, the thyroid hormone signaling pathway activator isan iodothyronine composition described in U.S. Pat. No. 7,163,918.

Epidermal Growth Factor (EGF) Family

Aspects of the disclosure relate to the use of growth factors from theEGF family as β cell maturation factors. It is contemplated that anygrowth factor from the EGF family that is capable, either alone or incombination with one or more other β cell maturation factors, ofinducing the differentiation of at least one insulin-producing,endocrine cell or precursor thereof into a SC-β cell can be used in themethods, compositions, and kits described herein. In some embodiments,the at least one growth factor from the EGF family comprisesbetacellulin. In some embodiments, at least one growth factor from theEGF family comprises EGF. Epidermal growth factor (EGF) is a 53 aminoacid cytokine which is proteolytically cleaved from a large integralmembrane protein precursor. In some embodiments, the growth factor fromthe EGF family comprises a variant EGF polypeptide, for example anisolated epidermal growth factor polypeptide having at least 90% aminoacid identity to the human wild-type EGF polypeptide sequence, asdisclosed in U.S. Pat. No. 7,084,246. In some embodiments, the growthfactor from the EGF family comprises an engineered EGF mutant that bindsto and agonizes the EGF receptor, as is disclosed in U.S. Pat. No.8,247,531. In some embodiments, the at least one growth factor from theEGF family is replaced with an agent that activates a signaling pathwayin the EGF family. In some embodiments, the growth factor from the EGFfamily comprises a compound that mimics EGF. In certain embodiments, themethods, compositions, and kits disclosed herein exclude a growth factorfrom the EGF family.

Protein Kinase Inhibitors

Aspects of the disclosure relate to the use of protein kinase inhibitorsas β cell maturation factors. It is contemplated that any protein kinaseinhibitor that is capable, either alone or in combination with other βcell maturation factors, of inducing the differentiation of at least oneinsulin-producing, endocrine cell or precursor thereof into a SC-β cellcan be used in the methods, compositions, and kits described herein.

In some embodiments, the protein kinase inhibitor comprisesstaurosporine. In some embodiments, the protein kinase inhibitorcomprises an analog of staurosporine. Exemplary analogs of staurosporineinclude, without limitation, Ro-31-8220, a bisindolylmaleimide (Bis)compound,10′-{5″-[(methoxycarbonyl)amino]-2″-methyl}-phenylaminocarbonylstaurosporine,a staralog (see, e.g., Lopez et al., “Staurosporine-derived inhibitorsbroaden the scope of analog-sensitive kinase technology”, J. Am. Chem.Soc. 2013; 135(48):18153-18159), and, cgp41251.

In some embodiments, the protein kinase inhibitor is an inhibitor ofPKCβ. In some embodiments, the protein kinase inhibitor is an inhibitorof PKCβ with the following structure or a derivative, analogue orvariant of the compound as follows:

In some embodiments, the inhibitor of PKCβ is a GSK-2 compound with thefollowing structure or a derivative, analogue or variant of the compoundas follows:

In some embodiments, the inhibitor of PKC is a bisindolylmaleimide.Exemplary bisindolylmaleimides include, without limitation,bisindolylmaleimide I, bisindolylmaleimide II, bisindolylmaleimide III,hydrochloride, or a derivative, analogue or variant. In someembodiments, a derivative or variant or analogue thereof is selectedfrom a derivative or variant of analogue of a compound selected from thecompounds selected from:

In some embodiments, the PKC inhibitor is a pseudohypericin, or aderivative, analogue or variant of the compound as follows:

In some embodiments, the PKC inhibitor is indorublin-3-monoxime, 5-Iodoor a derivative, analogue or variant of the following compound:

In certain embodiments, the methods, compositions, and kits disclosedherein exclude a protein kinase inhibitor.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Inhibitor

Aspects of the disclosure relate to the use of CFTR inhibitors as β cellmaturation factors. It is contemplated that any CFTR inhibitor that iscapable, either alone or in combination with one or more other β cellmaturation factors, of inducing the differentiation of at least oneinsulin-producing, endocrine cell or precursor thereof into a SC-β cellcan be used in the methods, compositions, and kits described herein.

Numerous CFTR inhibitors of use herein are available to the skilledartisan. Exemplary CFTR inhibitors include, without limitation, LPA2receptor agonist inhibitors of CFTR disclosed in U.S. Pub. No.2007/0078111, hydrazide-containing CFTR inhibitors disclosed in U.S.Pat. No. 7,888,332, and CFTR inhibitors disclosed in WIPO Pub. No.WO/2008/121877, a CFTR inhibitor compound disclosed in U.S. Pub. No.2008/0269206. In some embodiments, the CFTR inhibitor comprises aglycine hydrazide pore-occluding CFTR inhibitor. In some embodiments,the CFTR inhibitor comprises Gly-H101. In some embodiments, the CFTRinhibitor comprises a Gly-H101 derivative or analog (see, e.g.,Muanprasat et al., “Discovery of Glycine Hydrazide Pore-occluding CFTRInhibitors”, J. Gen. Physiol 2004; 124(2):125-137). In certainembodiments, the methods, compositions, and kits disclosed hereinexclude a CFTR inhibitor.

O-GlcNAcase Inhibitor

Aspects of the disclosure relate to the use of O-GlcNAcase inhibitors asβ cell maturation factors. It is contemplated that any O-GlcNAcaseinhibitor that is capable, either alone or in combination with one ormore other β cell maturation factors, of inducing the differentiation ofat least one insulin-producing, endocrine cell or precursor thereof intoa SC-β cell can be used in the methods, compositions, and kits describedherein. Numerous O-GlcNAcase inhibitors of use herein are available tothe skilled artisan. Exemplary O-GlcNAcase inhibitors include, withoutlimitation, permeable glycosidase inhibitors (see, e.g., WIPO Pub. No.WO/2013/169576 and WO/2013/166654), and selective glycosidase inhibitors(see, e.g., WIPO Pub. No. WO/2013/000084 and WO/2013/000085). In someembodiments, the O-GlcNAcase inhibitor comprises Thiamet G. In certainembodiments, the methods, compositions, and kits disclosed hereinexclude a O-GlcNAcase inhibitor.

Admixture Compositions

Another aspect of the present invention relates to an admixture ofinsulin-positive endocrine cells or precursor thereof, and at least oneβ cell maturation factor, for example, for inducing the differentiationof at least one insulin-positive endocrine cell or precursor thereof tobecome SC-β cells.

In another aspect of the present invention relates to composition, suchas a reaction admixture comprising at least one insulin-positiveendocrine cell or precursor thereof (e.g. a population ofinsulin-positive endocrine cells or precursors thereof fordifferentiating into SC-β cells) and at least one β cell maturationfactor. Alternatively, the present invention relates to a reactionadmixture comprising (i) a population of SC-β cells produced by chemicalinduction of differentiation of a population of insulin-positiveendocrine cells or precursors thereof to a SC-β cell, and (ii) at leastone β cell maturation factor.

In some embodiments, the concentrations of the at least one β cellmaturation factor added to the reaction mixture is a sufficient dose forinducing at least one insulin-positive endocrine cells or precursorsthereof to differentiate to a SC-β cell, as described herein.

In some embodiments, the composition comprises a concentration of atleast one β cell maturation factor of about between 25 nM to 10 μM, orbetween about 25 nM to 50 nM, or about 50 nM to 100 nM, or about 100 nMto 200 nM, or about 200 nM to about 500 nM or about 500 nM to about 1μM, or about 1 μM to 2 m, or about 2 μM to 5 m, or about 5 μM to 10 μM.

In some embodiments, a composition or admixture comprises aconcentration of at least one β cell maturation factor of at least about5 nM, at least about 7 nM, at least about 10 nM, at least about 12 nM,at least about 15 nM, at least about 17 nM, at least about 20 nM, atleast about 25 nM, at least about 30 nM, at least about 35 nM, at leastabout 40 nM, at least about 45 nM, at least about 50 nM, at least about100 nM or at least about 200 nM, or at least about 300 nM or at leastabout 400 nM or at least about 500 nM or more than 500 nM, or anyinteger between 10-500 nM or any integer between 5-50 nM, or any integerbetween 50-100 nM, or any integer between 100 nM-200 nM or any integerbetween 200 nM-500 nM. In some embodiments, a composition or admixturecomprises a concentration of at least one β cell maturation factor of atleast about 0.1 μM, or at least about 0.2 μM, or at least about 0.3 μM,or at least about 0.4 μM, or at least about 0.5 μM, or at least about 1μM, at least about 1.5 μM, at least about 2 μM, at least about 2.5 μM,at least about 3 μM, at least about 3.5 μM, at least about 4 μM, atleast about 4.5 μM, at least about 5 μM, at least about 6 μM, at leastabout 7 μM, at least about 8 μM, at least about 9 μM, or at least about10 μM, or more than 10 μM, or any integer between 0.1-0. μM or anyinteger between about 0.5-1 μM or any integer between 0.1-10 μM, or anyinteger between 0.5-5 μM, or any integer between 5 μM-10 μM.

Compositions and Kits

Described herein are compositions which comprise a cell described herein(e.g., a SC-β cell or mature pancreatic β cell). In some embodiments,the composition also includes a β cell maturation factor describedherein and/or cell culture media. Described herein are also compositionscomprising the compounds described herein (e.g. cell culture mediacomprising one or more of the compounds described herein). Describedherein are kits.

Another aspect of the present invention relates to kits for practicingmethods disclosed herein and for making SC-β cells or mature pancreaticβ cells disclosed herein. In one aspect, a kit includes at least oneinsulin-positive endocrine cell or precursor thereof and at least one βcell maturation factor as described herein, and optionally, the kit canfurther comprise instructions for converting at least oneinsulin-positive endocrine cell or precursor thereof to a population ofSC-β cells using a method described herein. In some embodiments, the kitcomprises at least two β cell maturation factors. In some embodiments,the kit comprises at least three β cell maturation factors. In someembodiments, the kit comprises at least four β cell maturation factors.In some embodiments, the kit comprises at least five β cell maturationfactors. In some embodiments, the kit comprises at least six β cellmaturation factors. In some embodiments, the kit comprises at leastseven β cell maturation factors. In some embodiments, the kit comprisesat least eight β cell maturation factors. In some embodiments, the kitcomprises at least nine β cell maturation factors. In some embodiments,the kit comprises at least ten β cell maturation factors. In someembodiments, the kit comprises β cell maturation factors fordifferentiating pluripotent cells to definitive endoderm cells. In someembodiments, the kit comprises β cell maturation factors fordifferentiating definitive endoderm cells to primitive gut tube cells.In some embodiments, the kit comprises R cell maturation factors fordifferentiating primitive gut tube cells to Pdx1-positive pancreaticprogenitor cells. In some embodiments, the kit comprises β cellmaturation factors for differentiating NKX6-1-positive pancreaticprogenitor cells to insulin-positive endocrine cells. In someembodiments, the kit comprises β cell maturation factors fordifferentiating insulin-positive endocrine cells to SC-β cells.

In some embodiments, the kit comprises any combination of β cellmaturation factors, e.g., for differentiating pluripotent cells todefinitive endoderm cells, differentiating definitive endoderm cells toprimitive gut tube cells, differentiating primitive gut tube cells toPdx1-positive pancreatic progenitor cells, differentiatingNKX6-1-positive pancreatic progenitor cells to insulin-positiveendocrine cells, and differentiating insulin-positive endocrine cells toSC-β cells.

In one embodiment, the kit can comprise a pluripotent stem cell for thepurposes of being used as a positive control, for example to assess ormonitor the effectiveness or ability of a compound of formula (I) tochemically induce the pluripotent stem cell to differentiate into atleast one insulin-positive endocrine cell or precursors thereof, andsubsequently into a SC-β cell. Accordingly, the kit can comprisesufficient amount of at least one β cell maturation factor for inducingthe differentiation of a control pluripotent stem cell population(positive control) as well as inducing the differentiation of apopulation of pluripotent stem cells of interest (e.g. the userspreferred pluripotent stem cell e.g. an iPS cell) into at least oneinsulin-positive endocrine cell or precursors thereof, or into a SC-βcell.

In some embodiment, the compound in the kit can be provided in awatertight or gas tight container which in some embodiments issubstantially free of other components of the kit. The compound can besupplied in more than one container, e.g., it can be supplied in acontainer having sufficient reagent for a predetermined number ofreactions e.g., 1, 2, 3 or greater number of separate reactions toinduce pluripotent stem cells to definitive endoderm cells, andsubsequently into insulin-positive endocrine cells or precursorsthereof, and subsequently into SC-β cells. A cell maturation factor canbe provided in any form, e.g., liquid, dried or lyophilized form. It ispreferred that a compound(s) (e.g., β cell maturation factors) describedherein be substantially pure and/or sterile. When a compound(s)described herein is provided in a liquid solution, the liquid solutionpreferably is an aqueous solution, with a sterile aqueous solution beingpreferred. When a compound(s) described herein is provided as a driedform, reconstitution generally is by the addition of a suitable solvent.The solvent, e.g., sterile water or buffer, can optionally be providedin the kit.

In some embodiments, the kit further optionally comprises informationmaterial. The informational material can be descriptive, instructional,marketing or other material that relates to the methods described hereinand/or the use of a compound(s) described herein for the methodsdescribed herein.

The informational material of the kits is not limited in its instructionor informative material. In one embodiment, the informational materialcan include information about production of the compound, molecularweight of the compound, concentration, date of expiration, batch orproduction site information, and so forth. In one embodiment, theinformational material relates to methods for administering thecompound. Additionally, the informational material of the kits is notlimited in its form. In many cases, the informational material, e.g.,instructions, is provided in printed matter, e.g., a printed text,drawing, and/or photograph, e.g., a label or printed sheet. However, theinformational material can also be provided in other formats, such asBraille, computer readable material, video recording, or audiorecording. In another embodiment, the informational material of the kitis contact information, e.g., a physical address, email address,website, or telephone number, where a user of the kit can obtainsubstantive information about a compound described herein and/or its usein the methods described herein. Of course, the informational materialcan also be provided in any combination of formats.

In one embodiment, the informational material can include instructionsto administer a compound(s) (e.g., a β cell maturation factor) asdescribed herein in a suitable manner to perform the methods describedherein, e.g., in a suitable dose, dosage form, or mode of administration(e.g., a dose, dosage form, or mode of administration described herein)(e.g., to a cell in vitro or a cell in vivo). In another embodiment, theinformational material can include instructions to administer acompound(s) described herein to a suitable subject, e.g., a human, e.g.,a human having or at risk for a disorder described herein or to a cellin vitro.

In addition to a compound(s) described herein, the composition of thekit can include other ingredients, such as a solvent or buffer, astabilizer, a preservative, a flavoring agent (e.g., a bitter antagonistor a sweetener), a fragrance or other cosmetic ingredient, and/or anadditional agent, e.g., for inducing pluripotent stem cells (e.g., invitro) or for treating a condition or disorder described herein.Alternatively, the other ingredients can be included in the kit, but indifferent compositions or containers than a compound described herein.In such embodiments, the kit can include instructions for admixing acompound(s) described herein and the other ingredients, or for using acompound(s) described herein together with the other ingredients, e.g.,instructions on combining the two agents prior to administration.

A β cell maturation factor as described herein can be provided in anyform, e.g., liquid, dried or lyophilized form. It is preferred that acompound(s) described herein be substantially pure and/or sterile. Whena compound(s) described herein is provided in a liquid solution, theliquid solution preferably is an aqueous solution, with a sterileaqueous solution being preferred. When a compound(s) described herein isprovided as a dried form, reconstitution generally is by the addition ofa suitable solvent. The solvent, e.g., sterile water or buffer, canoptionally be provided in the kit.

The kit can include one or more containers for the compositioncontaining at least one β cell maturation factor as described herein. Insome embodiments, the kit contains separate containers (e.g., twoseparate containers for the two agents), dividers or compartments forthe composition(s) and informational material. For example, thecomposition can be contained in a bottle, vial, or syringe, and theinformational material can be contained in a plastic sleeve or packet.In other embodiments, the separate elements of the kit are containedwithin a single, undivided container. For example, the composition iscontained in a bottle, vial or syringe that has attached thereto theinformational material in the form of a label. In some embodiments, thekit includes a plurality (e.g., a pack) of individual containers, eachcontaining one or more unit dosage forms (e.g., a dosage form describedherein) of a compound described herein. For example, the kit includes aplurality of syringes, ampules, foil packets, or blister packs, eachcontaining a single unit dose of a compound described herein. Thecontainers of the kits can be air tight, waterproof (e.g., impermeableto changes in moisture or evaporation), and/or light-tight.

The kit optionally includes a device suitable for administration of thecomposition, e.g., a syringe, inhalant, pipette, forceps, measuredspoon, dropper (e.g., eye dropper), swab (e.g., a cotton swab or woodenswab), or any such delivery device. In a preferred embodiment, thedevice is a medical implant device, e.g., packaged for surgicalinsertion.

The kit can also include a component for the detection of a marker forSC-β cells, e.g., for a marker described herein, e.g., a reagent for thedetection of positive SC-β cells. Or in some embodiments, the kit canalso comprise reagents for the detection of negative markers of SC-βcells for the purposes of negative selection of SC-β cells or foridentification of cells which do not express these negative markers(e.g., SC-β cells). The reagents can be, for example, an antibodyagainst the marker or primers for a RT-PCR or PCR reaction, e.g., asemi-quantitative or quantitative RT-PCR or PCR reaction. Such markerscan be used to evaluate whether an iPS cell has been produced. If thedetection reagent is an antibody, it can be supplied in dry preparation,e.g., lyophilized, or in a solution. The antibody or other detectionreagent can be linked to a label, e.g., a radiological, fluorescent(e.g., GFP) or colorimetric label for use in detection. If the detectionreagent is a primer, it can be supplied in dry preparation, e.g.,lyophilized, or in a solution.

It may be desirable to perform an analysis of the karyotype of the SC-βcells. Accordingly, the kit can include a component for karyotyping,e.g., a probe, a dye, a substrate, an enzyme, an antibody or otheruseful reagents for preparing a karyotype from a cell.

The kit can include SC-β cells, e.g., mature pancreatic β cells derivedfrom the same type of insulin-positive endocrine cell or precursorthereof, for example for the use as a positive cell type control.

The kit can also include informational materials, e.g., instructions,for use of two or more of the components included in the kit.

The informational material can be descriptive, instructional, marketingor other material that relates to the methods described herein and/orthe use of a compound(s) described herein for differentiating apluripotent stem cell according to the methods described herein. In oneembodiment, the informational material can include information aboutproduction of the compound, molecular weight of the compound,concentration, date of expiration, batch or production site information,and so forth. In one embodiment, the informational material relates tomethods for culturing a population of insulin-positive endocrine cellsin the presence of at least one β cell maturation factor describedherein.

Methods of Administering a Cell

In one embodiment, the cells described herein, e.g. a population ofSC-cells are transplantable, e.g., a population of SC-β cells can beadministered to a subject. In some embodiment, the subject who isadministered a population of SC-β cells is the same subject from whom apluripotent stem cell used to differentiate into a SC-β cell wasobtained (e.g. for autologous cell therapy). In some embodiments, thesubject is a different subject. In some embodiments, a subject sufferingfrom diabetes such as type I diabetes, or is a normal subject. Forexample, the cells for transplantation (e.g. a composition comprising apopulation of SC-β cells) can be a form suitable for transplantation,e.g., organ transplantation.

The method can further include administering the cells to a subject inneed thereof, e.g., a mammalian subject, e.g., a human subject. Thesource of the cells can be a mammal, preferably a human. The source orrecipient of the cells can also be a non-human subject, e.g., an animalmodel. The term “mammal” includes organisms, which include mice, rats,cows, sheep, pigs, rabbits, goats, horses, monkeys, dogs, cats, andpreferably humans. Likewise, transplantable cells can be obtained fromany of these organisms, including a non-human transgenic organism. Inone embodiment, the transplantable cells are genetically engineered,e.g., the cells include an exogenous gene or have been geneticallyengineered to inactivate or alter an endogenous gene.

A composition comprising a population of SC-β cells can be administeredto a subject using an implantable device. Implantable devices andrelated technology are known in the art and are useful as deliverysystems where a continuous, or timed-release delivery of compounds orcompositions delineated herein is desired. Additionally, the implantabledevice delivery system is useful for targeting specific points ofcompound or composition delivery (e.g., localized sites, organs). Negrinet al., Biomaterials, 22(6):563 (2001). Timed-release technologyinvolving alternate delivery methods can also be used in this invention.For example, timed-release formulations based on polymer technologies,sustained-release techniques and encapsulation techniques (e.g.,polymeric, liposomal) can also be used for delivery of the compounds andcompositions delineated herein.

Pharmaceutical Compositions Comprising a Population ofInsulin-Producing, Glucose Responsive Cells

For administration to a subject, a cell population produced by themethods as disclosed herein, e.g. a population of SC-β cells (producedby contacting at least one insulin-positive endocrine cell with at leastone β cell maturation factor (e.g., any one, two, three, four, five, ormore β cell maturation factors as described herein) can be administeredto a subject, for example in pharmaceutically acceptable compositions.These pharmaceutically acceptable compositions comprise atherapeutically-effective amount a population of SC-β cells as describedabove, formulated together with one or more pharmaceutically acceptablecarriers (additives) and/or diluents.

As described in detail below, the pharmaceutical compositions of thepresent invention can be specially formulated for administration insolid or liquid form, including those adapted for the following: (1)oral administration, for example, drenches (aqueous or non-aqueoussolutions or suspensions), lozenges, dragees, capsules, pills, tablets(e.g., those targeted for buccal, sublingual, and systemic absorption),boluses, powders, granules, pastes for application to the tongue; (2)parenteral administration, for example, by subcutaneous, intramuscular,intravenous or epidural injection as, for example, a sterile solution orsuspension, or sustained-release formulation; (3) topical application,for example, as a cream, ointment, or a controlled-release patch orspray applied to the skin; (4) intravaginally or intrarectally, forexample, as a pessary, cream or foam; (5) sublingually; (6) ocularly;(7) transdermally; (8) transmucosally; or (9) nasally. Additionally,compounds can be implanted into a patient or injected using a drugdelivery system. See, for example, Urquhart, et al., Ann. Rev.Pharmacol. Toxicol. 24: 199-236 (1984); Lewis, ed. “Controlled Releaseof Pesticides and Pharmaceuticals” (Plenum Press, New York, 1981); U.S.Pat. No. 3,773,919; and U.S. Pat. No. 35 3,270,960.

As used here, the term “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used here, the term “pharmaceutically-acceptable carrier” means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. Some examples of materials which canserve as pharmaceutically-acceptable carriers include: (1) sugars, suchas lactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, methylcellulose, ethyl cellulose,microcrystalline cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such asmagnesium stearate, sodium lauryl sulfate and talc; (8) excipients, suchas cocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C₂-C₁₂ alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.The terms such as “excipient”, “carrier”, “pharmaceutically acceptablecarrier” or the like are used interchangeably herein.

The phrase “therapeutically-effective amount” as used herein in respectto a population of cells means that amount of relevant cells in apopulation of cells, e.g., SC-β cells or mature pancreatic β cells, orcomposition comprising SC-β cells of the present invention which iseffective for producing some desired therapeutic effect in at least asub-population of cells in an animal at a reasonable benefit/risk ratioapplicable to any medical treatment. For example, an amount of apopulation of SC-β cells administered to a subject that is sufficient toproduce a statistically significant, measurable change in at least onesymptom of Type 1, Type 1.5 or Type 2 diabetes, such as glycosylatedhemoglobin level, fasting blood glucose level, hypoinsulinemia, etc.Determination of a therapeutically effective amount is well within thecapability of those skilled in the art. Generally, a therapeuticallyeffective amount can vary with the subject's history, age, condition,sex, as well as the severity and type of the medical condition in thesubject, and administration of other pharmaceutically active agents.

As used herein, the term “administer” refers to the placement of acomposition into a subject by a method or route which results in atleast partial localization of the composition at a desired site suchthat desired effect is produced. A compound or composition describedherein can be administered by any appropriate route known in the artincluding, but not limited to, oral or parenteral routes, includingintravenous, intramuscular, subcutaneous, transdermal, airway (aerosol),pulmonary, nasal, rectal, and topical (including buccal and sublingual)administration.

Exemplary modes of administration include, but are not limited to,injection, infusion, instillation, inhalation, or ingestion. “Injection”includes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intraventricular, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal,intracerebro spinal, and intrasternal injection and infusion. Inpreferred embodiments, the compositions are administered by intravenousinfusion or injection.

By “treatment”, “prevention” or “amelioration” of a disease or disorderis meant delaying or preventing the onset of such a disease or disorder,reversing, alleviating, ameliorating, inhibiting, slowing down orstopping the progression, aggravation or deterioration the progressionor severity of a condition associated with such a disease or disorder.In one embodiment, the symptoms of a disease or disorder are alleviatedby at least 5%, at least 10%, at least 20%, at least 30%, at least 40%,or at least 50%.

Treatment of Diabetes is determined by standard medical methods. A goalof Diabetes treatment is to bring sugar levels down to as close tonormal as is safely possible. Commonly set goals are 80-120 milligramsper deciliter (mg/dl) before meals and 100-140 mg/dl at bedtime. Aparticular physician may set different targets for the patent, dependingon other factors, such as how often the patient has low blood sugarreactions. Useful medical tests include tests on the patient's blood andurine to determine blood sugar level, tests for glycosylated hemoglobinlevel (HbA1c; a measure of average blood glucose levels over the past2-3 months, normal range being 4-6%), tests for cholesterol and fatlevels, and tests for urine protein level. Such tests are standard testsknown to those of skill in the art (see, for example, American DiabetesAssociation, 1998). A successful treatment program can also bedetermined by having fewer patients in the program with complicationsrelating to Diabetes, such as diseases of the eye, kidney disease, ornerve disease.

Delaying the onset of diabetes in a subject refers to delay of onset ofat least one symptom of diabetes, e.g., hyperglycemia, hypoinsulinemia,diabetic retinopathy, diabetic nephropathy, blindness, memory loss,renal failure, cardiovascular disease (including coronary arterydisease, peripheral artery disease, cerebrovascular disease,atherosclerosis, and hypertension), neuropathy, autonomic dysfunction,hyperglycemic hyperosmolar coma, or combinations thereof, for at least 1week, at least 2 weeks, at least 1 month, at least 2 months, at least 6months, at least 1 year, at least 2 years, at least 5 years, at least 10years, at least 20 years, at least 30 years, at least 40 years or more,and can include the entire lifespan of the subject.

In certain embodiments, the subject is a mammal, e.g., a primate, e.g.,a human. The terms, “patient” and “subject” are used interchangeablyherein. Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but are notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of Type 1diabetes, Type 2 Diabetes Mellitus, or pre-diabetic conditions. Inaddition, the methods described herein can be used to treat domesticatedanimals and/or pets. A subject can be male or female. A subject can beone who has been previously diagnosed with or identified as sufferingfrom or having Diabetes (e.g., Type 1 or Type 2), one or morecomplications related to Diabetes, or a pre-diabetic condition, andoptionally, but need not have already undergone treatment for theDiabetes, the one or more complications related to Diabetes, or thepre-diabetic condition. A subject can also be one who is not sufferingfrom Diabetes or a pre-diabetic condition. A subject can also be one whohas been diagnosed with or identified as suffering from Diabetes, one ormore complications related to Diabetes, or a pre-diabetic condition, butwho show improvements in known Diabetes risk factors as a result ofreceiving one or more treatments for Diabetes, one or more complicationsrelated to Diabetes, or the pre-diabetic condition. Alternatively, asubject can also be one who has not been previously diagnosed as havingDiabetes, one or more complications related to Diabetes, or apre-diabetic condition. For example, a subject can be one who exhibitsone or more risk factors for Diabetes, complications related toDiabetes, or a pre-diabetic condition, or a subject who does not exhibitDiabetes risk factors, or a subject who is asymptomatic for Diabetes,one or more Diabetes-related complications, or a pre-diabetic condition.A subject can also be one who is suffering from or at risk of developingDiabetes or a pre-diabetic condition. A subject can also be one who hasbeen diagnosed with or identified as having one or more complicationsrelated to Diabetes or a pre-diabetic condition as defined herein, oralternatively, a subject can be one who has not been previouslydiagnosed with or identified as having one or more complications relatedto Diabetes or a pre-diabetic condition.

As used herein, the phrase “subject in need of SC-β cells” refers to asubject who is diagnosed with or identified as suffering from, having orat risk for developing diabetes (e.g., Type 1, Type 1.5 or Type 2), oneor more complications related to diabetes, or a pre-diabetic condition.

A subject in need of a population of SC-β cells can be identified usingany method used for diagnosis of diabetes. For example, Type 1 diabetescan be diagnosed using a glycosylated hemoglobin (A1C) test, a randomblood glucose test and/or a fasting blood glucose test. Parameters fordiagnosis of diabetes are known in the art and available to skilledartisan without much effort.

In some embodiments, the methods of the invention further compriseselecting a subject identified as being in need of additional SC-βcells. A subject in need a population of SC-β cells can be selectedbased on the symptoms presented, such as symptoms of type 1, type 1.5 ortype 2 diabetes. Exemplary symptoms of diabetes include, but are notlimited to, excessive thirst (polydipsia), frequent urination(polyuria), extreme hunger (polyphagia), extreme fatigue, weight loss,hyperglycemia, low levels of insulin, high blood sugar (e.g., sugarlevels over 250 mg, over 300 mg), presence of ketones present in urine,fatigue, dry and/or itchy skin, blurred vision, slow healing cuts orsores, more infections than usual, numbness and tingling in feet,diabetic retinopathy, diabetic nephropathy, blindness, memory loss,renal failure, cardiovascular disease (including coronary arterydisease, peripheral artery disease, cerebrovascular disease,atherosclerosis, and hypertension), neuropathy, autonomic dysfunction,hyperglycemic hyperosmolar coma, and combinations thereof.

In some embodiments, a composition comprising a population of SC-β cellsfor administration to a subject can further comprise a pharmaceuticallyactive agent, such as those agents known in the art for treatment ofdiabetes and or for having anti-hyperglycemic activities, for example,inhibitors of dipeptidyl peptidase 4 (DPP-4) (e.g., Alogliptin,Linagliptin, Saxagliptin, Sitagliptin, Vildagliptin, and Berberine),biguanides (e.g., Metformin, Buformin and Phenformin), peroxisomeproliferator-activated receptor (PPAR) modulators such asthiazolidinediones (TZDs) (e.g., Pioglitazone, Rivoglitazone,Rosiglitazone and Troglitazone), dual PPAR agonists (e.g., Aleglitazar,Muraglitazar and Tesaglitazar), sulfonylureas (e.g., Acetohexamide,Carbutamide, Chlorpropamide, Gliclazide, Tolbutamide, Tolazamide,Glibenclamide (Glyburide), Glipizide, Gliquidone, Glyclopyramide, andGlimepiride), meglitinides (“glinides”) (e.g., Nateglinide, Repaglinideand Mitiglinide), glucagon-like peptide-1 (GLP-1) and analogs (e.g.,Exendin-4, Exenatide, Liraglutide, Albiglutide), insulin and insulinanalogs (e.g., Insulin lispro, Insulin aspart, Insluin glulisine,Insulin glargine, Insulin detemir, Exubera and NPH insulin),alpha-glucosidase inhibitors (e.g., Acarbose, Miglitol and Voglibose),amylin analogs (e.g. Pramlintide), Sodium-dependent glucosecotransporter T2 (SGLT T2) inhibitors (e.g., Dapgliflozin, Remogliflozinand Sergliflozin) and others (e.g. Benfluorex and Tolrestat).

In type 1 diabetes, β cells are undesirably destroyed by continuedautoimmune response. Thus, this autoimmune response can be attenuated byuse of compounds that inhibit or block such an autoimmune response. Insome embodiments, a composition comprising a population of SC-β cellsfor administration to a subject can further comprise a pharmaceuticallyactive agent which is a immune response modulator. As used herein, theterm “immune response modulator” refers to compound (e.g., asmall-molecule, antibody, peptide, nucleic acid, or gene therapyreagent) that inhibits autoimmune response in a subject. Without wishingto be bound by theory, an immune response modulator inhibits theautoimmune response by inhibiting the activity, activation, orexpression of inflammatory cytokines (e.g., IL-12, IL-23 or IL-27), orSTAT-4. Exemplary immune response modulators include, but are notlimited to, members of the group consisting of Lisofylline (LSF) and theLSF analogs and derivatives described in U.S. Pat. No. 6,774,130,contents of which are herein incorporated by reference in theirentirety.

A composition comprising SC-β cells can be administrated to the subjectin the same time, of different times as the administration of apharmaceutically active agent or composition comprising the same. Whenadministrated at different times, the compositions comprising apopulation of SC-β cells and/or pharmaceutically active agent foradministration to a subject can be administered within 5 minutes, 10minutes, 20 minutes, 60 minutes, 2 hours, 3 hours, 4, hours, 8 hours, 12hours, 24 hours of administration of the other. When a compositionscomprising a population of SC-β cells and a composition comprising apharmaceutically active agent are administered in differentpharmaceutical compositions, routes of administration can be different.In some embodiments, a subject is administered a composition comprisingSC-β cells. In other embodiments, a subject is administered acomposition comprising a pharmaceutically active agent. In anotherembodiment, a subject is administered a compositions comprising apopulation of SC-β cells mixed with a pharmaceutically active agent. Inanother embodiment, a subject is administered a composition comprising apopulation of SC-β cells and a composition comprising a pharmaceuticallyactive agent, where administration is substantially at the same time, orsubsequent to each other.

Toxicity and therapeutic efficacy of administration of a compositionscomprising a population of SC-β cells can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). Compositions comprising a population of SC-β cells thatexhibit large therapeutic indices, are preferred.

The amount of a composition comprising a population of SC-β cells can betested using several well-established animal models.

The non-obese diabetic (NOD) mouse carries a genetic defect that resultsin insulitis showing at several weeks of age (Yoshida et al., Rev.Immunogenet. 2:140, 2000). 60-90% of the females develop overt diabetesby 20-30 weeks. The immune-related pathology appears to be similar tothat in human Type I diabetes. Other models of Type I diabetes are micewith transgene and knockout mutations (Wong et al., Immunol. Rev.169:93, 1999). A rat model for spontaneous Type I diabetes was recentlyreported by Lenzen et al. (Diabetologia 44:1189, 2001). Hyperglycemiacan also be induced in mice (>500 mg glucose/dL) by way of a singleintraperitoneal injection of streptozotocin (Soria et al., Diabetes49:157, 2000), or by sequential low doses of streptozotocin (Ito et al.,Environ. Toxicol. Pharmacol. 9:71, 2001). To test the efficacy ofimplanted islet cells, the mice are monitored for return of glucose tonormal levels (<200 mg/dL).

Larger animals provide a good model for following the sequelae ofchronic hyperglycemia. Dogs can be rendered insulin-dependent byremoving the pancreas (J. Endocrinol. 158:49, 2001), or by feedinggalactose (Kador et al., Arch. Opthalmol. 113:352, 1995). There is alsoan inherited model for Type I diabetes in keeshond dogs (Am. J. Pathol.105:194, 1981). Early work with a dog model (Banting et al., Can. Med.Assoc. J. 22:141, 1922) resulted in a couple of Canadians making a longocean journey to Stockholm in February of 1925.

Byway of illustration, a pilot study can be conducted by implanting apopulation of SC-β cells into the following animals: a) non-diabeticnude (T-cell deficient) mice; b) nude mice rendered diabetic bystreptozotocin treatment; and c) nude mice in the process ofregenerating islets following partial pancreatectomy. The number ofcells transplanted is equivalent to ^(˜)1000-2000 normal human β cellsimplanted under the kidney capsule, in the liver, or in the pancreas.For non-diabetic mice, the endpoints of can be assessment of graftsurvival (histological examination) and determination of insulinproduction by biochemical analysis, RIA, ELISA, andimmunohistochemistry. Streptozotocin treated and partiallypancreatectomized animals can also be evaluated for survival, metaboliccontrol (blood glucose) and weight gain.

In some embodiments, data obtained from the cell culture assays and inanimal studies can be used in formulating a range of dosage for use inhumans. The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized.

The therapeutically effective dose of a composition comprising apopulation of SC-β cells can also be estimated initially from cellculture assays. A dose may be formulated in animal models in vivo toachieve a secretion of insulin at a concentration which is appropriatein response to circulating glucose in the plasma. Alternatively, theeffects of any particular dosage can be monitored by a suitablebioassay.

With respect to duration and frequency of treatment, it is typical forskilled clinicians to monitor subjects in order to determine when thetreatment is providing therapeutic benefit, and to determine whether toincrease or decrease dosage, increase or decrease administrationfrequency, discontinue treatment, resume treatment or make otheralteration to treatment regimen. The dosing schedule can vary from oncea week to daily depending on a number of clinical factors, such as thesubject's sensitivity to the polypeptides. The desired dose can beadministered at one time or divided into subdoses, e.g., 2-4 subdosesand administered over a period of time, e.g., at appropriate intervalsthrough the day or other appropriate schedule. Such sub-doses can beadministered as unit dosage forms. In some embodiments, administrationis chronic, e.g., one or more doses daily over a period of weeks ormonths. Examples of dosing schedules are administration daily, twicedaily, three times daily or four or more times daily over a period of 1week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months,5 months, or 6 months or more.

In another aspect of the invention, the methods provide use of anisolated population of SC-β cells as disclosed herein. In one embodimentof the invention, an isolated population of SC-β cells as disclosedherein may be used for the production of a pharmaceutical composition,for the use in transplantation into subjects in need of treatment, e.g.a subject that has, or is at risk of developing diabetes, for examplebut not limited to subjects with congenital and acquired diabetes. Inone embodiment, an isolated population of SC-β cells may be geneticallymodified. In another aspect, the subject may have or be at risk ofdiabetes and/or metabolic disorder. In some embodiments, an isolatedpopulation of SC-β cells as disclosed herein may be autologous and/orallogeneic. In some embodiments, the subject is a mammal, and in otherembodiments the mammal is a human.

The use of an isolated population of SC-β cells as disclosed hereinprovides advantages over existing methods because the population of SC-βcells can be differentiated from insulin-positive endocrine cells orprecursors thereof derived from stem cells, e.g. iPS cells obtained orharvested from the subject administered an isolated population of SC-βcells. This is highly advantageous as it provides a renewable source ofSC-β cells with can be differentiated from stem cells toinsulin-positive endocrine cells by methods commonly known by one ofordinary skill in the art, and then further differentiated by themethods described herein to pancreatic β-like cells or cells withpancreatic β cell characteristics, for transplantation into a subject,in particular a substantially pure population of mature pancreaticβ-like cells that do not have the risks and limitations of cells derivedfrom other systems.

In another embodiment, an isolated population of SC-β cells (e.g.,mature pancreatic β cells or β-like cells can be used as models forstudying properties for the differentiation into insulin-producingcells, e.g. to pancreatic β cells or pancreatic β-like cells, orpathways of development of cells of endoderm origin into pancreatic βcells.

In some embodiments, the insulin-positive endocrine cells or SC-β cellsmay be genetically engineered to comprise markers operatively linked topromoters that are expressed when a marker is expressed or secreted, forexample, a marker can be operatively linked to an insulin promoter, sothat the marker is expressed when the insulin-positive endocrine cellsor precursors thereof differentiation into SC-β cells which express andsecrete insulin. In some embodiments, a population of SC-β cells can beused as a model for studying the differentiation pathway of cells whichdifferentiate into islet β cells or pancreatic β-like cells.

In other embodiments, the insulin-producing, glucose responsive cellscan be used as models for studying the role of islet β cells in thepancreas and in the development of diabetes and metabolic disorders. Insome embodiments, the SC-β cells can be from a normal subject, or from asubject which carries a mutation and/or polymorphism (e.g. in the genePdx1 which leads to early-onset insulin-dependent diabetes mellitus(NIDDM), as well as maturity onset diabetes of the young type 4 (MODY4),which can be used to identify small molecules and other therapeuticagents that can be used to treat subjects with diabetes with a mutationor polymorphism in Pdx1. In some embodiments, the SC-β cells may begenetically engineered to correct the polymorphism in the Pdx1 geneprior to being administered to a subject in the therapeutic treatment ofa subject with diabetes. In some embodiments, the SC-β cells may begenetically engineered to carry a mutation and/or polymorphism.

In one embodiment of the invention relates to a method of treatingdiabetes or a metabolic disorder in a subject comprising administeringan effective amount of a composition comprising a population of SC-βcells as disclosed herein to a subject with diabetes and/or a metabolicdisorder. In a further embodiment, the invention provides a method fortreating diabetes, comprising administering a composition comprising apopulation of SC-β cells as disclosed herein to a subject that has, orhas increased risk of developing diabetes in an effective amountsufficient to produce insulin in response to increased blood glucoselevels.

In one embodiment of the above methods, the subject is a human and apopulation of SC-β cells as disclosed herein are human cells. In someembodiments, the invention contemplates that a population of SC-β cellsas disclosed herein are administered directly to the pancreas of asubject, or is administered systemically. In some embodiments, apopulation of SC-β cells as disclosed herein can be administered to anysuitable location in the subject, for example in a capsule in the bloodvessel or the liver or any suitable site where administered thepopulation of SC-β cells can secrete insulin in response to increasedglucose levels in the subject.

The present invention is also directed to a method of treating a subjectwith diabetes or a metabolic disorder which occurs as a consequence ofgenetic defect, physical injury, environmental insult or conditioning,bad health, obesity and other diabetes risk factors commonly known by aperson of ordinary skill in the art. Efficacy of treatment of a subjectadministered a composition comprising a population of SC-β cells can bemonitored by clinically accepted criteria and tests, which include forexample, (i) Glycated hemoglobin (A1C) test, which indicates a subjectsaverage blood sugar level for the past two to three months, by measuringthe percentage of blood sugar attached to hemoglobin, theoxygen-carrying protein in red blood cells. The higher your blood sugarlevels, the more hemoglobin has sugar attached. An A1C level of 6.5percent or higher on two separate tests indicates the subject hasdiabetes. A test value of 6-6.5% suggest the subject has prediabetes.(ii) Random blood sugar test. A blood sample will be taken from thesubject at a random time, and a random blood sugar level of 200milligrams per deciliter (mg/dL)-11.1 millimoles per liter (mmol/L), orhigher indicated the subject has diabetes. (iii) Fasting blood sugartest. A blood sample is taken from the subject after an overnight fast.A fasting blood sugar level between 70 and 99 mg/dL (3.9 and 5.5 mmol/L)is normal. If the subjects fasting blood sugar levels is 126 mg/dL (7mmol/L) or higher on two separate tests, the subject has diabetes. Ablood sugar level from 100 to 125 mg/dL (5.6 to 6.9 mmol/L) indicatesthe subject has prediabetes. (iv) Oral glucose tolerance test. A bloodsample will be taken after the subject has fasted for at least eighthours or overnight and then ingested a sugary solution, and the bloodsugar level will be measured two hours later. A blood sugar level lessthan 140 mg/dL (7.8 mmol/L) is normal. A blood sugar level from 140 to199 mg/dL (7.8 to 11 mmol/L) is considered prediabetes. This issometimes referred to as impaired glucose tolerance (IGT). A blood sugarlevel of 200 mg/dL (11.1 mmol/L) or higher may indicate diabetes.

In some embodiments, the effects of administration of a population ofSC-β cells as disclosed herein to a subject in need thereof isassociated with improved exercise tolerance or other quality of lifemeasures, and decreased mortality. The effects of cellular therapy witha population of SC-β cells can be evident over the course of days toweeks after the procedure. However, beneficial effects may be observedas early as several hours after the procedure, and may persist forseveral years. In some embodiments, the effects of cellular therapy witha population of SC-β cells occurs within two weeks after the procedure.

In some embodiments, a population of SC-β cells as disclosed herein maybe used for tissue reconstitution or regeneration in a human patient orother subject in need of such treatment. In some embodimentscompositions of populations of SC-β cells can be administered in amanner that permits them to graft or migrate to the intended tissue siteand reconstitute or regenerate the functionally deficient area. Specialdevices are available that are adapted for administering cells capableof reconstituting a population of β cells in the pancreas or at analternative desired location. Accordingly, the SC-β cells may beadministered to a recipient subject's pancreas by injection, oradministered by intramuscular injection.

In some embodiments, compositions comprising a population of SC-β cellsas disclosed herein have a variety of uses in clinical therapy,research, development, and commercial purposes. For therapeuticpurposes, for example, a population of SC-β cells as disclosed hereinmay be administered to enhance insulin production in response toincrease in blood glucose level for any perceived need, such as aninborn error in metabolic function, the effect of a disease condition(e.g. diabetes), or the result of significant trauma (i.e. damage to thepancreas or loss or damage to islet β cells). In some embodiments, apopulation of SC-β cells as disclosed herein are administered to thesubject not only help restore function to damaged or otherwise unhealthytissues, but also facilitate remodeling of the damaged tissues.

To determine the suitability of cell compositions for therapeuticadministration, the population of SC-β cells can first be tested in asuitable animal model. At one level, cells are assessed for theirability to survive and maintain their phenotype in vivo. Cellcompositions comprising SC-β cells can be administered toimmunodeficient animals (such as nude mice, or animals renderedimmunodeficient chemically or by irradiation). Tissues are harvestedafter a period of regrowth, and assessed as to whether the administeredcells or progeny thereof are still present.

This can be performed by administering cells that express a detectablelabel (such as green fluorescent protein, or β-galactosidase); that havebeen prelabeled (for example, with BrdU or [3H] thymidine), or bysubsequent detection of a constitutive cell marker (for example, usinghuman-specific antibody). The presence and phenotype of the administeredpopulation of SC-β cells can be assessed by immunohistochemistry orELISA using human-specific antibody, or by RT-PCR analysis using primersand hybridization conditions that cause amplification to be specific forhuman polynucleotides, according to published sequence data.

A number of animal models for testing diabetes are available for suchtesting, and are commonly known in the art, for example as disclosed inU.S. Pat. No. 6,187,991 which is incorporated herein by reference, aswell as rodent models; NOD (non-obese mouse), BB_DB mice, KDP rat andTCR mice, and other animal models of diabetes as described in Rees etal, Diabet Med. 2005 April; 22(4):359-70; Srinivasan K, et al., Indian JMed. Res. 2007 March; 125(3):451-7; Chatzigeorgiou A, et al., In Vivo.2009 March-April; 23(2):245-58, which are incorporated herein byreference.

In some embodiments, a population of SC-β cells as disclosed herein maybe administered in any physiologically acceptable excipient, where theSC-β cells may find an appropriate site for replication, proliferation,and/or engraftment. In some embodiments, a population of SC-β cells asdisclosed herein can be introduced by injection, catheter, or the like.In some embodiments, a population of SC-β cells as disclosed herein canbe frozen at liquid nitrogen temperatures and stored for long periods oftime, being capable of use on thawing. If frozen, a population of SC-βcells will usually be stored in a 10% DMSO, 50% FCS, 40% RPMI 1640medium. Once thawed, the cells may be expanded by use of growth factorsand/or feeder cells associated with culturing SC-β cells as disclosedherein.

In some embodiments, a population of SC-β cells as disclosed herein canbe supplied in the form of a pharmaceutical composition, comprising anisotonic excipient prepared under sufficiently sterile conditions forhuman administration. For general principles in medicinal formulation,the reader is referred to Cell Therapy: Stem Cell Transplantation, GeneTherapy, and Cellular Immunotherapy, by G. Morstyn & W. Sheridan eds,Cambridge University Press, 1996; and Hematopoietic Stem Cell Therapy,E. D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000. Choice ofthe cellular excipient and any accompanying elements of the compositioncomprising a population of SC-β cells as disclosed herein will beadapted in accordance with the route and device used for administration.In some embodiments, a composition comprising a population of SC-β cellscan also comprise or be accompanied with one or more other ingredientsthat facilitate the engraftment or functional mobilization of the SC-βcells. Suitable ingredients include matrix proteins that support orpromote adhesion of the SC-β cells, or complementary cell types,especially endothelial cells. In another embodiment, the composition maycomprise resorbable or biodegradable matrix scaffolds.

In some embodiments, a population of SC-β cells as disclosed herein maybe genetically altered in order to introduce genes useful ininsulin-producing cells such as pancreatic β cells, e.g. repair of agenetic defect in an individual, selectable marker, etc., or genesuseful in selection against non-insulin-producing cells differentiatedfrom at least one insulin-positive endocrine or precursor thereof or forthe selective suicide of implanted SC-β cells. In some embodiments, apopulation of SC-β cells can also be genetically modified to enhancesurvival, control proliferation, and the like. In some embodiments apopulation of SC-β cells as disclosed herein can be genetically alteringby transfection or transduction with a suitable vector, homologousrecombination, or other appropriate technique, so that they express agene of interest. In one embodiment, a population of SC-β cells istransfected with genes encoding a telomerase catalytic component (TERT),typically under a heterologous promoter that increases telomeraseexpression beyond what occurs under the endogenous promoter, (seeInternational Patent Application WO 98/14592, which is incorporatedherein by reference). In other embodiments, a selectable marker isintroduced, to provide for greater purity of the population of SC-βcells. In some embodiments, a population of SC-β cells may begenetically altered using vector containing supernatants over a 8-16 hperiod, and then exchanged into growth medium for 1-2 days. Geneticallyaltered SC-β cells can be selected using a drug selection agent such aspuromycin, G418, or blasticidin, and then recultured.

Gene therapy can be used to either modify a cell to replace a geneproduct, to facilitate regeneration of tissue, to treat disease, or toimprove survival of the cells following implantation into a subject(i.e. prevent rejection).

In an alternative embodiment, a population of SC-β cells as disclosedherein can also be genetically altered in order to enhance their abilityto be involved in tissue regeneration, or to deliver a therapeutic geneto a site of administration. A vector is designed using the knownencoding sequence for the desired gene, operatively linked to a promoterthat is either pan-specific or specifically active in the differentiatedcell type. Of particular interest are cells that are genetically alteredto express one or more growth factors of various types, such assomatostatin, glucagon, and other factors.

Many vectors useful for transferring exogenous genes into target SC-βcells as disclosed herein are available. The vectors may be episomal,e.g. plasmids, virus derived vectors such as cytomegalovirus,adenovirus, etc., or may be integrated into the target cell genome,through homologous recombination or random integration, e.g. retrovirusderived vectors such MMLV, HIV-1, ALV, etc. In some embodiments,combinations of retroviruses and an appropriate packaging cell line mayalso find use, where the capsid proteins will be functional forinfecting the SC-β cells as disclosed herein. Usually, SC-β cells andvirus will be incubated for at least about 24 hours in the culturemedium. In some embodiments, the SC-β cells are then allowed to grow inthe culture medium for short intervals in some applications, e.g. 24-73hours, or for at least two weeks, and may be allowed to grow for fiveweeks or more, before analysis. Commonly used retroviral vectors are“defective”, i.e. unable to produce viral proteins required forproductive infection. Replication of the vector requires growth in thepackaging cell line.

The host cell specificity of the retrovirus is determined by theenvelope protein, env (p120). The envelope protein is provided by thepackaging cell line. Envelope proteins are of at least three types,ecotropic, amphotropic and xenotropic. Retroviruses packaged withecotropic envelope protein, e.g. MMLV, are capable of infecting mostmurine and rat cell types. Ecotropic packaging cell lines include BOSC23(Pear et al. (1993) P.N.A.S. 90:8392-8396). Retroviruses bearingamphotropic envelope protein, e.g. 4070A (Danos et al, supra.), arecapable of infecting most mammalian cell types, including human, dog andmouse. Amphotropic packaging cell lines include PAl2 (Miller et al.(1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller et al. (1986) Mol.Cell. Biol. 6:2895-2902) GRIP (Danos et al. (1988) PNAS 85:6460-6464).Retroviruses packaged with xenotropic envelope protein, e.g. AKR env,are capable of infecting most mammalian cell types, except murine cells.In some embodiments, the vectors may include genes that must later beremoved, e.g. using a recombinase system such as Cre/Lox, or the cellsthat express them destroyed, e.g. by including genes that allowselective toxicity such as herpesvirus TK, Bcl-Xs, etc.

Suitable inducible promoters are activated in a desired target celltype, either the transfected cell, or progeny thereof. Bytranscriptional activation, it is intended that transcription will beincreased above basal levels in the target cell by at least about 100fold, more usually by at least about 1000 fold. Various promoters areknown that are induced in different cell types.

In one aspect of the present invention, a population of SC-β cells asdisclosed herein are suitable for administering systemically or to atarget anatomical site. A population of SC-β cells can be grafted intoor nearby a subject's pancreas, for example, or may be administeredsystemically, such as, but not limited to, intra-arterial or intravenousadministration. In alternative embodiments, a population of SC-β cellsof the present invention can be administered in various ways as would beappropriate to implant in the pancreatic or secretory system, includingbut not limited to parenteral, including intravenous and intraarterialadministration, intrathecal administration, intraventricularadministration, intraparenchymal, intracranial, intracisternal,intrastriatal, and intranigral administration. Optionally, a populationof SC-β cells are administered in conjunction with an immunosuppressiveagent.

In some embodiments, a population of SC-β cells can be administered anddosed in accordance with good medical practice, taking into account theclinical condition of the individual patient, the site and method ofadministration, scheduling of administration, patient age, sex, bodyweight and other factors known to medical practitioners. Thepharmaceutically “effective amount” for purposes herein is thusdetermined by such considerations as are known in the art. The amountmust be effective to achieve improvement, including but not limited toimproved survival rate or more rapid recovery, or improvement orelimination of symptoms and other indicators as are selected asappropriate measures by those skilled in the art. A population of SC-βcells can be administered to a subject the following locations: clinic,clinical office, emergency department, hospital ward, intensive careunit, operating room, catheterization suites, and radiologic suites.

In other embodiments, a population of SC-β cells is stored for laterimplantation/infusion. A population of SC-β cells may be divided intomore than one aliquot or unit such that part of a population of SC-βcells is retained for later application while part is appliedimmediately to the subject. Moderate to long-term storage of all or partof the cells in a cell bank is also within the scope of this invention,as disclosed in U.S. Patent Application Serial No. 20030054331 andPatent Application No. WO03024215, and is incorporated by reference intheir entireties. At the end of processing, the concentrated cells maybe loaded into a delivery device, such as a syringe, for placement intothe recipient by any means known to one of ordinary skill in the art.

In some embodiments a population of SC-β cells can be applied alone orin combination with other cells, tissue, tissue fragments, growthfactors such as VEGF and other known angiogenic or arteriogenic growthfactors, biologically active or inert compounds, resorbable plasticscaffolds, or other additive intended to enhance the delivery, efficacy,tolerability, or function of the population. In some embodiments, apopulation of SC-β cells may also be modified by insertion of DNA or byplacement in cell culture in such a way as to change, enhance, orsupplement the function of the cells for derivation of a structural ortherapeutic purpose. For example, gene transfer techniques for stemcells are known by persons of ordinary skill in the art, as disclosed in(Morizono et al., 2003; Mosca et al., 2000), and may include viraltransfection techniques, and more specifically, adeno-associated virusgene transfer techniques, as disclosed in (Walther and Stein, 2000) and(Athanasopoulos et al., 2000). Non-viral based techniques may also beperformed as disclosed in (Murarnatsu et al., 1998).

In another aspect, in some embodiments, a population of SC-β cells couldbe combined with a gene encoding pro-angiogenic growth factor(s). Genesencoding anti-apoptotic factors or agents could also be applied.Addition of the gene (or combination of genes) could be by anytechnology known in the art including but not limited to adenoviraltransduction, “gene guns,” liposome-mediated transduction, andretrovirus or lentivirus-mediated transduction, plasmid adeno-associatedvirus. Cells could be implanted along with a carrier material bearinggene delivery vehicle capable of releasing and/or presenting genes tothe cells over time such that transduction can continue or be initiated.Particularly when the cells and/or tissue containing the cells areadministered to a patient other than the patient from whom the cellsand/or tissue were obtained, one or more immunosuppressive agents may beadministered to the patient receiving the cells and/or tissue to reduce,and preferably prevent, rejection of the transplant. As used herein, theterm “immunosuppressive drug or agent” is intended to includepharmaceutical agents which inhibit or interfere with normal immunefunction. Examples of immunosuppressive agents suitable with the methodsdisclosed herein include agents that inhibit T-cell/B-cell costimulationpathways, such as agents that interfere with the coupling of T-cells andB-cells via the CTLA4 and B7 pathways, as disclosed in U.S. Patent Pub.No 2002/0182211, which is incorporated herein by reference. In oneembodiment, a immunosuppressive agent is cyclosporine A. Other examplesinclude myophenylate mofetil, rapamicin, and anti-thymocyte globulin. Inone embodiment, the immunosuppressive drug is administered with at leastone other therapeutic agent. The immunosuppressive drug is administeredin a formulation which is compatible with the route of administrationand is administered to a subject at a dosage sufficient to achieve thedesired therapeutic effect. In another embodiment, the immunosuppressivedrug is administered transiently for a sufficient time to inducetolerance to the cardiovascular stem cells of the invention.

Pharmaceutical compositions comprising effective amounts of a populationof SC-β cells are also contemplated by the present invention. Thesecompositions comprise an effective number of SC-β cells, optionally, incombination with a pharmaceutically acceptable carrier, additive orexcipient. In certain aspects of the present invention, a population ofSC-β cells are administered to the subject in need of a transplant insterile saline. In other aspects of the present invention, a populationof SC-β cells are administered in Hanks Balanced Salt Solution (HBSS) orIsolyte S, pH 7.4. Other approaches may also be used, including the useof serum free cellular media. In one embodiment, a population of SC-βcells are administered in plasma or fetal bovine serum, and DMSO.Systemic administration of a population of SC-β cells to the subject maybe preferred in certain indications, whereas direct administration atthe site of or in proximity to the diseased and/or damaged tissue may bepreferred in other indications.

In some embodiments, a population of SC-β cells can optionally bepackaged in a suitable container with written instructions for a desiredpurpose, such as the reconstitution or thawing (if frozen) of apopulation of SC-β cells prior to administration to a subject.

In one embodiment, an isolated population of SC-β cells as disclosedherein are administered with a differentiation agent. In one embodiment,the SC-β cells are combined with the differentiation agent toadministration into the subject. In another embodiment, the cells areadministered separately to the subject from the differentiation agent.Optionally, if the cells are administered separately from thedifferentiation agent, there is a temporal separation in theadministration of the cells and the differentiation agent. The temporalseparation may range from about less than a minute in time, to abouthours or days in time. The determination of the optimal timing and orderof administration is readily and routinely determined by one of ordinaryskill in the art.

Diagnosis of Diabetes

Type 1 diabetes is an autoimmune disease that results in destruction ofinsulin-producing β cells of the pancreas. Lack of insulin causes anincrease of fasting blood glucose (around 70-120 mg/dL in nondiabeticpeople) that begins to appear in the urine above the renal threshold(about 190-200 mg/dl in most people). The World Health Organizationdefines the diagnostic value of fasting plasma glucose concentration to7.0 mmol/l (126 mg/dl) and above for Diabetes Mellitus (whole blood 6.1mmol/l or 110 mg/dl), or 2-hour glucose level of 11.1 mmol/L or higher(200 mg/dL or higher).

Type 1 diabetes can be diagnosed using a variety of diagnostic teststhat include, but are not limited to, the following: (1) glycatedhemoglobin (A1C) test, (2) random blood glucose test and/or (3) fastingblood glucose test.

The Glycated hemoglobin (A1C) test is a blood test that reflects theaverage blood glucose level of a subject over the preceding two to threemonths. The test measures the percentage of blood glucose attached tohemoglobin, which correlates with blood glucose levels (e.g., the higherthe blood glucose levels, the more hemoglobin is glycosylated). An A1Clevel of 6.5 percent or higher on two separate tests is indicative ofdiabetes. A result between 6 and 6.5 percent is considered prediabetic,which indicates a high risk of developing diabetes.

The Random Blood Glucose Test comprises obtaining a blood sample at arandom time point from a subject suspected of having diabetes. Bloodglucose values can be expressed in milligrams per deciliter (mg/dL) ormillimoles per liter (mmol/L). A random blood glucose level of 200 mg/dL(11.1 mmol/L) or higher indicates the subject likely has diabetes,especially when coupled with any of the signs and symptoms of diabetes,such as frequent urination and extreme thirst.

For the fasting blood glucose test, a blood sample is obtained after anovernight fast. A fasting blood glucose level less than 100 mg/dL (5.6mmol/L) is considered normal. A fasting blood glucose level from 100 to125 mg/dL (5.6 to 6.9 mmol/L) is considered prediabetic, while a levelof 126 mg/dL (7 mmol/L) or higher on two separate tests is indicative ofdiabetes.

Type 1 diabetes can also be distinguished from type 2 diabetes using aC-peptide assay, which is a measure of endogenous insulin production.The presence of anti-islet antibodies (to Glutamic Acid Decarboxylase,Insulinoma Associated Peptide-2 or insulin), or lack of insulinresistance, determined by a glucose tolerance test, is also indicativeof type 1, as many type 2 diabetics continue to produce insulininternally, and all have some degree of insulin resistance.

Testing for GAD 65 antibodies has been proposed as an improved test fordifferentiating between type 1 and type 2 diabetes as it appears thatthe immune system is involved in Type 1 diabetes etiology.

In some embodiments, the present invention provides compositions for theuse of populations of SC-β cells produced by the methods as disclosedherein to restore islet function in a subject in need of such therapy.Any condition relating to inadequate production of a pancreaticendocrine (insulin, glucagon, or somatostatin), or the inability toproperly regulate secretion may be considered for treatment with cells(e.g. populations of SC-β cells) prepared according to this invention,as appropriate. Of especial interest is the treatment of Type I(insulin-dependent) diabetes mellitus.

Subjects in need thereof can be selected for treatment based onconfirmed long-term dependence on administration of exogenous insulin,and acceptable risk profile. The subject receives approximately 10,000SC-β cells or cell equivalents per kg body weight. If the cells are notautologouse, in order to overcome an allotype mismatch, the subject canbe treated before surgery with an immunosuppressive agent such as FK506and rapamycin (orally) and daclizumab (intravenously). A compositioncomprising a population of SC-β cells can be infused through a catheterin the portal vein. The subject can then be subjected to abdominalultrasound and blood tests to determine liver function. Daily insulinrequirement is tracked, and the subject is given a second transplant ifrequired. Follow-up monitoring includes frequent blood tests for druglevels, immune function, general health status, and whether the patientremains insulin independent.

General approaches to the management of the diabetic patient areprovided in standard textbooks, such as the Textbook of InternalMedicine, 3rd Edition, by W. N. Kelley ed., Lippincott-Raven, 1997; andin specialized references such as Diabetes Mellitus: A Fundamental andClinical Text 2nd Edition, by D. Leroith ed., Lippincott Williams &Wilkins 2000; Diabetes (Atlas of Clinical Endocrinology Vol. 2) by C. R.Kahn et al. eds., Blackwell Science 1999; and Medical Management of Type1 Diabetes 3rd Edition, McGraw Hill 1998. Use of islet cells for thetreatment of Type I diabetes is discussed at length in CellularInter-Relationships in the Pancreas: Implications for IsletTransplantation, by L. Rosenberg et al., Chapman & Hall 1999; and FetalIslet Transplantation, by C. M. Peterson et al. eds., Kluwer 1995.

As always, the ultimate responsibility for subject selection, the modeof administration, and dosage of a population of SC-β cells is theresponsibility of the managing clinician. For purposes of commercialdistribution, populations of SC-0 cells as disclosed herein aretypically supplied in the form of a pharmaceutical composition,comprising an isotonic excipient prepared under sufficiently sterileconditions for human administration. This invention also includes setsof populations of SC-β cells that exist at any time during theirmanufacture, distribution, or use. The sets of populations of SC-β cellscomprise any combination of two or more cell populations described inthis disclosure, exemplified but not limited to the differentiation ofdefinitive endoderm cells to become pdx1-positive pancreatic progenitorcells, and their subsequent differentiation e.g. into insulin-producingcells such as mature pancreatic β cells or mature pancreatic β-likecells as the term is defined herein. In some embodiments, the cellcompositions comprising populations of SC-β cells can be administered(e.g. implanted into a subject) in combination with other cell typese.g. other differentiated cell types, sometimes sharing the same genome.Each cell type in the set may be packaged together, or in separatecontainers in the same facility, or at different locations, undercontrol of the same entity or different entities sharing a businessrelationship.

For general principles in medicinal formulation of cell compositions,the reader is referred to Cell Therapy: Stem Cell Transplantation, GeneTherapy, and Cellular Immunotherapy, by G. Morstyn & W. Sheridan eds,Cambridge University Press, 1996. The composition is optionally packagedin a suitable container with written instructions for a desired purpose,such as the treatment of diabetes.

In some embodiments, compositions comprising populations of SC-β cellscan also be used as the functional component in a mechanical devicedesigned to produce one or more of the endocrine polypeptides ofpancreatic islet cells. In its simplest form, the device contains apopulation of SC-β cells behind a semipermeable membrane that preventspassage of the cell population, retaining them in the device, butpermits passage of insulin, glucagon, or somatostatin secreted by thecell population. This includes populations of SC-β cells that aremicroencapsulated, typically in the form of cell clusters to permit thecell interaction that inhibits dedifferentiation. For example, U.S. Pat.No. 4,391,909 describe islet cells encapsulated in a spheroidsemipermeable membrane made up of polysaccharide polymers >3,000 mol.wt. that are cross-linked so that it is permeable to proteins the sizeof insulin, but impermeable to molecules over 100,000 mol. wt. U.S. Pat.No. 6,023,009 describes islet cells encapsulated in a semipermeablemembrane made of agarose and agaropectin. Microcapsules of this natureare adapted for administration into the body cavity of a diabeticpatient, and are thought to have certain advantages in reducinghistocompatibility problems or susceptibility to bacteria.

More elaborate devices are also contemplated for use to comprise apopulation of SC-β cells, either for implantation into diabeticpatients, or for extracorporeal therapy. U.S. Pat. No. 4,378,016describes an artificial endocrine gland containing an extracorporealsegment, a subcutaneous segment, and a replaceable envelope containingthe hormone-producing cells. U.S. Pat. No. 5,674,289 describes abioartificial pancreas having an islet chamber, separated by asemipermeable membrane to one or more vascularizing chambers open tosurrounding tissue. Useful devices typically have a chamber adapted tocontain the islet cells, and a chamber separated from the islet cells bya semipermeable membrane which collects the secreted proteins from theislet cells, and which may also permit signaling back to the isletcells, for example, of the circulating glucose level.

Methods of Identifying β Cell Maturation Factors that Increase theProduction of SC-β cells or Pancreatic β Cells

Described herein is a method of identifying a β cell maturation factoror agent that increases the production of SC-β cells (e.g., maturepancreatic β cells). In certain examples, a high content and/or highthroughput screening method is provided. The method includes exposing atleast one insulin-positive endocrine cell or a precursor thereof to atleast one compound (e.g., a library compound or a compound describedherein) and determining if the compound increases the production of SC-βcells, e.g., mature pancreatic β cells from the at least oneinsulin-positive endocrine cell or the precursor thereof. A cell can beidentified as a SC-β cell (e.g., a mature pancreatic β cell) using oneor more of the markers described herein. In some examples, the at leastone insulin-positive endocrine cell or the precursor thereof may bedifferentiated prior to exposure to the library. In other examples, twoor more compounds may be used, either individually or together, in thescreening assay. In additional examples, the at least oneinsulin-positive endocrine cell or the precursor thereof may be placedin a multi-well plate, and a library of compounds may be screened byplacing the various members of the library in different wells of themulti-well plate. Such screening of libraries can rapidly identifycompounds that are capable of generating SC-β cells, e.g., maturepancreatic β cells, from the at least one insulin-positive endocrinecell or precursor thereof.

In some embodiments, the method further comprises isolating a populationof the SC-β cells, e.g., pancreatic β cells (e.g., wherein at least 5%,10%, 15%, 20%, 25%, 30%, 35%, 50%, 75% or greater of the subject celltype).

In some embodiments, the method further comprises implanting the SC-βcells produced by the methods as disclosed herein into a subject (e.g.,a subject having diabetes, e.g., type I, type II or Type 1.5 diabetes).In some embodiments, the SC-β cell is derived from a stem cell obtainedfrom a subject. In some embodiments, the SC-β cell is derived from astem cell from a donor different than the subject, e.g., a relative ofthe subject.

In one aspect, the invention features a SC-β cell, e.g., a maturepancreatic β cell made by a method described herein. In another aspect,the invention features a composition comprising a SC-β cell made by amethod described herein.

In another aspect, the invention features a kit comprising:insulin-positive endocrine cells or precursors thereof; at least one βcell maturation factor described herein; and instructions for using theinsulin-positive endocrine cells or precursors thereof and the at leastone β cell maturation factor to produce a SC-β cell (e.g., maturepancreatic β cell). In some embodiments, the kit further comprises: acomponent for the detection of a marker for a mature β cell, e.g., for amarker described herein, e.g., a reagent for the detection of a markerof β cell maturity, e.g., an antibody against the marker; and a maturepancreatic β cell, e.g., for use as a control.

In some embodiments, the kit further comprises: a component todifferentiate an endodermal cell, e.g., a definitive endodermal cell toa cell of a second cell type, e.g., at least one insulin-positiveendocrine cell or precursors thereof; and instructions for using theendodermal cell (e.g., the definitive endodermal cell) described hereinand the component to produce the cell of a second type, e.g., at leastone insulin-positive endocrine cell or precursors thereof. In someembodiments, the kit further comprises: a component for the detection ofa marker for the cell of the second cell type, e.g., for a markerdescribed herein, e.g., a reagent for the detection of Pdx1, e.g., anantibody against the marker; and a cell or the second cell type, e.g.,at least one insulin-positive endocrine cell or precursors thereof,e.g., for use as a control.

In one aspect, the invention features a method of facilitatingdifferentiation of insulin-positive endocrine cells or precursorsthereof to SC-β cells comprising providing at least one insulin-positivepancreatic endocrine cell or precursor thereof, and providing at leastone β cell maturation factor (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore cell maturation factors described herein) to differentiate the atleast one insulin-positive endocrine cell or precursor thereof to a SC-βcell (e.g., a mature pancreatic β cell), upon exposure of the stem cellto the at least one β cell maturation factor. In some embodiments, theat least one insulin-positive endocrine cell or precursor thereof isfrom a mammal. In some embodiments, the at least one insulin-positiveendocrine cell or precursor thereof is from mouse or human. In someembodiments, the at least one insulin-positive endocrine cell orprecursor thereof derived from culturing an embryonic stem cell (e.g., amammalian embryonic stem cell such as a mouse or human embryonic stemcell). In some embodiments, the at least one insulin-positive endocrinecell or precursor thereof derived from culturing an induced pluripotentstem cell (e.g., a mammalian iPs cell such as a mouse or human iPscell).

In some embodiments, a plurality of insulin-positive endocrine cells orprecursors thereof are differentiated into a plurality of maturepancreatic β cells or SC-β cells, for example, by contacting theplurality of insulin-positive endocrine cells or precursors thereof withat least one, at least two, at least three, or more of the β cellmaturation factors as described herein.

In some embodiments, the a plurality of insulin-positive endocrine cellsor precursors thereof are exposed to the β cell maturation factors, forabout 1, 2, 4, 6, 8, 10, 12, 14, 16, or more days. In some embodiments,the plurality of insulin-positive endocrine cells or precursors thereofare exposed to the β cell maturation factors at a concentration of about25 nM, 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 400 nM, 500 nM, 600 nM,700 nM, 800 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM or 10 μM. In someembodiments, the plurality of insulin-positive endocrine cells orprecursors thereof are exposed to the β cell maturation factors at aconcentration of about 250 nM, 400 nM, 500 nM, 600 nM, 700 nM, or 800nM. In some embodiments, greater than about 20%, 30%, 40%, 50%, 60%,70%, 80%, or 90% of the insulin-positive endocrine cells or precursorsthereof are differentiated into the mature pancreatic β cells or SC-βcells.

In some aspects, the disclosure provides artificial islets constructedusing the SC-β cells described herein. In some aspects, an artificialislet comprises one or more SC-β cells differentiated in vitro frompluripotent stem cells, e.g., according to a method described herein.

In some aspects, the disclosure provides an artificial pancreascomprising SC-β cells differentiated in vitro from pluripotent stemcells.

It is understood that the foregoing detailed description and thefollowing examples are illustrative only and are not to be taken aslimitations upon the scope of the invention. Various changes andmodifications to the disclosed embodiments, which will be apparent tothose of skill in the art, may be made without departing from the spiritand scope of the disclosure. Further, all patents, patent applications,and publications identified are expressly incorporated herein byreference for the purpose of describing and disclosing, for example, themethodologies described in such publications that might be used inconnection with the disclosure. These publications are provided solelyfor their disclosure prior to the filing date of the presentapplication. Nothing in this regard should be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention or for any other reason. All statements as tothe date or representation as to the contents of these documents arebased on the information available to the applicants and do notconstitute any admission as to the correctness of the dates or contentsof these documents.

EXAMPLES Example 1—Generation of Functional Pancreatic β Cells In Vitro

Summary

The generation of insulin-producing pancreatic β cells from stem cellsin vitro would provide an unprecedented cell source for drug discoveryand cell transplantation therapy in diabetes. However, insulin-producingcells previously generated from human pluripotent stem cells (hPSC) lackmany characteristics of bona fide β cells including function in vitroand/or in vivo. The work described herein demonstrates an exemplaryscalable differentiation protocol that generates SC-β cells from hPSC invitro. Surprisingly, and unexpectedly, these SC-β cells secrete amountsof insulin comparable to adult β cells in response to multiplesequential glucose challenges, flux Ca2+, express markers found in βcells, and package insulin into secretary granules. As a proof ofconcept, SC-β cells also respond to known diabetes drugs andproliferative cues in vitro. Furthermore, the SC-β cells secrete highlevels of human insulin in the serum of mice immediately aftertransplantation, and transplantation of these cells immediatelyameliorates hyperglycemia in diabetic mice. The work described hereinrepresents a major advance in the use of stem cell-derived β cells(i.e., SC-β cells) for the treatment of diabetes and for in vitro β cellstudy and drug screening.

The following work demonstrates several advantages of the SC-β cellsproduced according to the methods described herein, for example, theSC-β cells perform glucose stimulated insulin secretion in vitro,resemble human islet β cells by gene expression and ultrastructure,secrete human insulin and ameliorate hyperglycemia when transplantedinto mice, provide a new platform for cell therapy (e.g.,transplantation into a subject in need of additional and/or functional βcells), drug screening (e.g., for insulin production/secretion,survival, dedifferentiation, etc.), research (e.g., determining thedifferences in function between normal and diabetic β cells), and tissueengineering (e.g., using the SC-β cells as the first cell type inreconstructing an islet).

Introduction

The discovery of human pluripotent stem cells (hPSC) opened the door tothe possibility that replacement cells and tissues could one day begenerated for disease treatment or drug screening. Research in the pastdecade has moved the field closer to that goal through development ofstrategies to generate cells that would otherwise be difficult toobtain, like neurons or cardiomyocytes (Kriks et al., 2011; Shiba etal., 2012). These cells have also been transplanted into animal modelsand are able to engraft into the host, in some cases with a beneficialeffect like suppression of arrhythmias with stem cell-derivedcardiomyocytes (Shiba et al., 2012), restoration of locomotion afterspinal injury with oligodendrocyte progenitor cells (Keirstead et al.,2005), or improved vision after transplantation of retinal epithelialcells into rodent models of blindness (Lu et al., 2009).

One of the most rapidly growing diseases that may be treatable by stemcell derived tissues is diabetes, affecting more than 300 million peopleworldwide according to the International Diabetes Federation. Type 1diabetes results from the autoimmune destruction of β cells in thepancreatic islet whereas the more common type 2 diabetes results fromperipheral tissue insulin resistance and β cell dysfunction. Thesepatients, particularly those suffering from type 1 diabetes, couldpotentially be cured through transplantation of new, functional β cells.Transplantation of cadaveric human islets has demonstrated that patientscan be made insulin independent for five year or longer via thisstrategy, but this approach is very limited because of the scarcity ofdonor human islets (Bellin et al., 2012). The generation of an unlimitedsupply of human β cells from stem cells could extend this therapy tomillions of new patients. β cells are an ideal test case forregenerative medicine as only a single cell type needs to be generatedand those cells can be placed anywhere in the body within animmunoprotective device (e.g., a microcapsule as described herein) as ormaterial with access to the vasculature.

Pharmaceutical screening to identify new drugs that can improve β cellfunction or proliferation is also hindered by limited supplies ofcadaveric islets and their high variability due to variation in cause ofdeath, donor genetic background, and other factors in their isolation.Thus a consistent, uniform supply of SC-β cells could provide a uniqueand valuable drug discovery platform for diabetes.

Research to date has made considerable progress towards generating the βcell lineage in vitro from hPSC. Definitive endoderm and subsequentpancreatic progenitors can now be differentiated with high efficiencies(Kroon et al., 2008; D'Amour et al., 2006; D'Amour et al., 2005; Rezaniaet al., 2012). Importantly these cells can further differentiate intofunctional β cells within three to four months after transplantationinto rodents (Kroon et al., 2008; Rezania et al., 2012), indicating thatthey contain the developmental potential to develop into β cells ifprovided enough time and appropriate cues. Unfortunately, themonths-long process the cells undergo in vivo remains a black box, andit is unclear if this process would work in human patients. Other workhas focused on generating insulin-producing cells from human pancreaticprogenitors in vitro. However, the cells generated to date are not bonafide β cells. These cells either fail to perform glucose stimulatedinsulin secretion in vitro, fail to express appropriate β cell markerslike NKX6-1 or PDX1, abnormally co-express other hormones like glucagon,fail to function after transplantation in vivo, or display a combinationof these abnormal features (D'Amour et al., 2006; Cheng et al., 2012;Narayanan et al., 2013; Xie et al., 2013; Nostro et al., 2011).

The work described herein provides a strategy for virtually unlimited,large-scale production of functional human β cells from hPSC in vitro.By using sequential modulation of signaling pathways in combination in a3-dimensional cell culture system, monohormonal insulin-producing cells(SC-β cells) that co-express key β cell markers and display β cellultrastructural features can be generated. Furthermore, these cellsmimic the function of human islets both in vitro and in vivo. Finally,the cells demonstrate proof of concept of their utility for the dualaims of in vitro drug screening and in vivo transplantation therapy fordiabetes.

Results

Generation of Glucose Sensing Insulin Secreting β Cells from hPSC InVitro

An exemplary method for generating functional β cells from hPSC in vitrois outlined in FIG. 1A. To produce large numbers of β cells, a scalablesuspension-based culture system that can generate >10⁸ hPSC and laterdifferentiated cell types was utilized (Schulz et al., 2012). Clustersof HUES8 human embryonic stem cells, approximately 100-200 μm indiameter, were induced into highly pure definitive endoderm (>95%Sox17+) and subsequently early pancreatic progenitors (>90% PDX1+) usingprotocols adapted from previous publications (FIG. 1B) (Schulz et al.,2012; Rezania et al., 2012). Next, a method using extended time inculture with FGF family member KGF, hedgehog inhibitor Sant1, and a lowconcentration of retinoic acid to generate high levels of NKX6-1+/PDX1+co-expressing pancreatic progenitor clusters (>60% NKX6-1+/PDX1+ cells)was identified (FIG. 1A.) Transplantation of these pancreaticprogenitors into mice has been reported to give rise to functional βcells in vivo after 3-4 months (Rezania et al., 2012). This was used asa starting point for developing the protocol to recapitulate thisgeneration of functional β cells in vitro.

The NKX6-1+/PDX1+ pancreatic progenitor cells were then differentiatedinto C-peptide-expressing endocrine cells using either a previouslypublished protocol (control differentiation) or a newly developedprotocol (new differentiation). The control differentiation protocolproduced cells over the course of several months that were monohormonalINS+ and INS+/GCG+ or INS+/SST+ polyhormonal (PH) cells. Thenomenclature PH was used to refer to this cell population. The newdifferentiation protocol, on the other hand, involved 2-3 weeks of aunique series of culture steps involving hedgehog signaling inhibition,retinoic acid signaling, gamma-secretase inhibition, TGFβ signalinginhibition, EGF signaling, thyroid hormone signaling and the islet mediaCMRL 1066 (Nostro et al., 2011; Rezania et al., 2012; Thowfeequ et al.,2007; Aguayo-Mazzucato et al., 2013; D'Amour et al., 2006). It washypothesized that the C-peptide+ cells generated with this newdifferentiation protocol were similar to primary adult β (1° β) cellsand, as such, are referred to stem cell-β (SC-β) cells (i.e., SC-βcells).

The key functional feature of a β cell is its ability to repeatedlyperform glucose stimulated insulin secretion (GSIS). Nearly all existingdirected differentiation protocols generate insulin-expressing cellsfrom hPSC that fail to perform GSIS in vitro (D'Amour et al., 2006). Oneprotocol has been reported using endodermal progenitor lines as astarting population that can make cells that could secrete some insulinin response to a single glucose challenge (Cheng et al., 2012).Conversely, the SC-β cells generated utilizing the methods describedherein can respond to at least three sequential high glucose challenges.These cells secreted high levels of insulin in a pattern similar toprimary adult β cells, while PH cells from the control protocol failedto respond to glucose (FIG. 2A-2C and FIG. 3A-2C). The stimulationindex, as calculated by the ratio of insulin secreted in high glucose(20 mM) to low glucose (2 mM), was similar for SC-β cells compared toprimary adult β cells, 2.3±0.9 and 2.3±1.4 respectively. Additionally,there was a small percentage of high glucose challenges for which bothSC-β cells and primary adult β cells both did not respond. Furthermore,the amount of insulin secreted per cell in response to 20 mM glucose bySC-β cells was indistinguishable from that secreted by primary adultβcells, 2.6±1.6 and 2.5±1.2 μIU/10³ cells, respectively. Taken together,this data suggests that the in vitro function of SC-β cells generatedusing the new differentiation protocol is very similar to their bonafide primary adult β cell counterparts.

The in vitro functionality of SC-β cells generated using the newdifferentiation protocol was later confirmed by measuring changes inintracellular Ca²⁺. β cells sense changing glucose levels throughcalcium signaling; increasing glucose levels leads to membranedepolarization causing an influx of calcium ions which is responsiblefor triggering insulin release (Mohammed et al., 2009). Thus calciuminflux in cellular clusters stained with Fluo-4 AM, a fluorescentcalcium indicator dye, in real-time using fluorescent microscopy wasmonitored (FIG. 4A). This method allowed analysis of calcium flux onboth a population and single cell level, and showed that both SC-β cellsand primary adult β cells responded to sequential glucose challenges byrepeatedly increasing intracellular Ca²⁺ in similar manners, consistentwith normal GSIS, while PH cells generated with the controldifferentiation protocol displayed an abnormal calcium response,consistent with their abnormal GSIS (FIG. 4B). When single cell analysiswas performed, most individual SC-β cells and primary adult β cellsresponded to 2-3 sequential glucose challenges by fluxing calcium whilemost PH cells responded to 0 challenges (FIG. 4C-4E). Unlike rodent βcells, human β cells are known to display a degree of dyssynchrony inresponse to high glucose (Rutter and Hodson, 2013). These data show thatboth the entire population and individual cells within the SC-β cellclusters function similarly to β cells within isolated islets andfurther support the conclusion that the SC-β cells generated using thenew differentiation protocol function in vitro.

Stem Cell-Derived f Cells from hPSC Resemble Primary Human β Cells

After observing that SC-β cells function like primary adult β cells invitro, the two cell populations were then analyzed by proteinexpression, gene expression, and ultrastructure. Unlike most previouslyreported hPSC-derived insulin-producing cells, these SC-β cells expressboth normal β cell markers PDX1 and NKX6-1 (FIGS. 5A and 5B). Rare non-βcell hormones are observed but do not co-localize with NKX6-1/C-peptideco-positive cells (FIG. 5C). SC-β cells stain positive for both insulinand C-peptide, a stoichiometric byproduct of proinsulin processing,indicating that the insulin staining comes from cell-endogenous insulinproduction (FIG. 6) and stain for ISL1, MAFA, and MAFB (FIG. 7A-7C).Flow cytometry quantification reveals that the methods described hereincan produce 40% NKX6-1/C-peptide, similar to the percentage found incadaveric human islets (FIG. 5D). Furthermore, only 8% of totalC-peptide+ cells co-express glucagon and 4% co-express somatostatin(FIG. 8A-8C.) Although largely monohormonal cells have been previouslyreported in one study, those cells were not shown to express key β cellidentity marker NKX6-1 or to function in vivo (Cheng 2012.) Recent workhas shown that directed differentiation protocols that generate higherlevels of NKX6-1 lead to better transplantation outcomes for thepancreatic progenitor transplants (Rezania et al., 2013). Additionally,conditional knock-out studies have shown that NKX6-1 expression isnecessary for β cell function in adult mouse islets, suggesting thatco-expression of these factors in our cells may help explain theirfunctional abilities (Taylor et al., 2013).

The improved protein expression of several key β cell markers indicatedthat the transcriptional network of these cells better matched that ofhuman islet β cells. Recent work has demonstrated that INS+ PH cellsgenerated by previous protocols do not resemble adult islet INS+ β cells(Hrvatin et al., 2014; Xie et al., 2013). Microarray analysis of sortedINS+ cells generated by previously published protocols showed that theyclustered with fetal β cells rather than with functional adult human βcells sorted via the same method.

To compare the SC-β cells of the disclosure to adult human islets,INS+/NKX6-1+ cells were sorted using the same method and performedglobal gene expression analysis by microarray. Unlike the previouslypublished stem cell-derived INS+ PH cells, the SC-β cells describedherein clustered more closely with human adult β cells than fetal βcells or INS+PH cells (FIG. 5E). In addition, these data showed that theexpression of canonical β cells genes, like PDX1, MNX1, and NKX2-2 weremore similar between SC-β cells and adult human β cells than previousINS+ PH cells. An analysis of the top 100 most differentially expressedgenes in the data set also showed that SC-β cells and adult human βcells were more similar to one another than previous PH or fetal β cells(FIG. 5F). There remain differences between SC-β cells and human adult βcells that could be improved with additional minor changes to theculture media composition during late stages of differentiation.

Since the gene and protein expression patterns of SC-β cells resemblethose of primary human β cells, it was hypothesized that SC-β cellsshould also package their insulin protein into appropriate secretorygranules like bona fide β cells do. β cells package insulin intosecretory granules that are initially pale grey with a halo and furthermature into dark crystallized polygonal granules with a light halo (FIG.9A). Previous studies of INS+ cells generated from stem cells revealedthat these cells have only polyhormonal and alpha-like granules, whichare distinct round granules with a dark halo (D'Amour et al., 2006;Kroon et al., 2008). These results were recapitulated with the controlprotocol that produced INS+ cells that had only abnormal polyhormonaland alpha-like granules and few, if any, β cell granules (FIG. 9A). Onthe other hand, the methods described herein generated SC-β cells thatpackaged and crystallized insulin protein into prototypical β cellgranules (FIG. 9A). Both developing insulin granules and mature,crystallized insulin granules were observed in both primary human βcells and SC-β cells (FIG. 9B). To confirm the protein content of thesegranules, immunogold labeling was performed with particles againstinsulin and glucagon. Whereas the PH cell granules abnormally containedboth insulin and glucagon protein, the primary human β cell and SC-βcells granules contained only insulin (FIG. 9C). Thus the ultrastructureof SC-β cells mirrors that of adult human β cells.

Generation of Glucose Sensing Insulin Secreting β Cells from hiPSC InVitro

The new differential protocol used to develop the functional β Cellsfrom hPSC as discussed above was further used to generate functional βcells from hiPSC lines in vitro. The hiPSC lines were generated at theHarvard iPSC Core with skin fibroblasts grown out from eithernon-diabetic or type 1 diabetic patients. In contrast to the hPSC linewhich was generated from embryos, the hiPSC line was generated fromhuman tissue, showing that the functional β cells can be developeddirectly from diseased patients, i.e., patients with type 1 diabetes.

The resulting β cells were subjected to glucose challenges to determinetheir ability to repeatedly perform glucose stimulated insulin secretion(GSIS). It was found that multiple β cells generated utilizing themethods described herein can respond to at least three sequential highglucose challenges (FIGS. 10A-10B). In this particular experiment,hiPSC-β cells derived from a non-diabetic cell line (1013-3FA) werecompared to hiPSC-β cells derived from a type 1 diabetic cell line(1031SeVA). The stimulation index, as calculated by the ratio of insulinsecreted in high glucose (20 mM) to low glucose (2 mM), was greater forthe β cells derived from the non-diabetic cell line compared to the βcells derived from the type 1 diabetic cell line. Furthermore, theamount of insulin secreted per cell in response to either a 2 mM or 20mM glucose challenge was greater for the β cells derived from the type 1diabetic cell line than for the β cells derived from the non-diabeticcell line. Taken together, these data suggest that the in vitro functionof hiPSC-β cells derived from a type 1 diabetic cell line is similar tothe non-diabetic cell counterparts.

After observing that hiPSC-β cells are responsive to glucose in vitro,the inventors next analyzed how similar the non-diabetic and type 1diabetic cell populations were by protein expression, gene expression,and ultrastructure. Three different non-diabetic cell lines and threedifferent type 1 diabetic cell lines were used to determine expressionof NKX6-1/C-peptide. As was the case with the hPSC, these hiPSC-β cellsexpressed both normal β cell markers PDX1 and NKX6-1 (FIGS. 11A-11F).Flow cytometry quantification reveals that the methods described hereincan produce about 40% NKX6-1/C-peptide, similar to the percentage foundin cadaveric human islets (FIG. 5D).

Stem Cell-Derived β Cells Function In Vivo after Transplantation

The data described thus far are consistent with the generation offunctional human β cells in vitro. To further test their capacity tofunction in vivo, these cells were transplanted under the kidney capsuleof immunocompromised mice (FIG. 12A-12D and Table S1).

TABLE S1 ELISA measurements of human insulin from the serum of micetransplanted with SC-β cells, primary β cells, and PH cells HumanInsulin Human Insulin Human Insulin (μIU/mL) (μIU/mL) (μIU/mL) Cell Typems# 0′ 30′ Cell Type ms# 0′ 30′ Cell Type ms# 0′ 30′ SC-β cells  1 2.2 1.1 1′ β cells  1 1.1  2.8 PH cells  1 nd 0.4  2 5.1  2.5  2 1.4  2.0 2 nd 0.1  3 1.8  4.9  3 0.5  2.4  3 nd 0.1  4 1.3  3.2  4 0.7  1.4  4nd 0.4  5 2.1  2.4  5 1.1  1.7  5 nd 0.4  6 nd  8.5  6 −0.4 −0.1  6 0.10.1  7 nd  7.5  7 1.4  3.9  7 0.2 0.2  8 nd  5.9  8 nd  5.1 *  8 0.0 0.3 9 nd  5.9  9 nd 13.0 *  9 0.0 −0.2 10 nd  2.9 10 nd  5.1 * 10 0.5 1.211 1.4  5.3 11 nd  1.5 * 11 nd 0.7 12 1.7  2.6 12 nd  4.4 12 nd 1.0 132.3  4.3 13 nd  7.0 13 nd 1.6 14 0.9  1.4 14 nd  2.2 14 nd 1.1 15 nd 0.3 15 nd  3.2 15 0.3 0.6 16 nd  3.5 16 nd  3.9 16 0.1 0.2 17 nd  4.317 1.0  0.6 17 2.6 0.1 18 nd  3.2 18 0.9  1.7 19 nd 45.0 # 19 5.9  2.620 nd 36.1 # 20 1.6  2.3 21 nd 14.8 # 21 1.0  1.7 22 nd 67.5 # 23 nd57.7 # 24 nd  3.0 25 nd  2.8 26 nd  5.6 27 nd  7.5 28 nd  7.8 29 nd  6.430 5.0 11.4 31 4.3  5.1 32 3.8  2.5 nd = not determined # cultured 2 wkin vitro during final step; all other SC-β cells cultured 1 wk

When adult human islets are transplanted, human insulin is detectable inthe serum of these mice within two weeks. Conversely, when previouslypublished pancreatic progenitors were transplanted into mice no insulinis detected at two weeks post-transplant (Kroon et al., 2008; Schulz etal., 2012; Rezania et al., 2012). Instead the cells require a 3-4 monthlong, poorly understood maturation phase in vivo. On the other hand,SC-β cells, like human islets, secrete high levels of insulin into thehost bloodstream in response to a glucose challenge within two weeks oftransplant (FIG. 12A). As a control, we also transplanted PH cellsgenerated using previously published protocols and found that thesecells did not secrete significant levels of insulin in response toglucose in vivo (FIG. 12A). Moreover, it was also confirmed that thepancreatic progenitors generated failed to produce appreciable insulinin vivo within two weeks, as has been previously published (FIG. 12C).

Some insulin producing cells that are not bona fide β cells can secreteinsulin in an unregulated manner into the bloodstream of animal,functioning like an insulin pellet rather than a responsive β cell. Totest whether SC-β cells not only secrete high levels of insulin but alsodo so in a functionally responsive manner the amount of human insulin inthe bloodstream of the mice both before and after an acute glucosechallenge was measured. For both human islet transplants and SC-β cellstransplants, 9 out of 12 mice tested showed functional glucosestimulated insulin secretion in vivo two weeks post-transplant (FIG.12A).

After the terminal in vivo GSIS challenge was performed, these animalswere sacrificed and the engrafted kidneys removed for analysis.Histological sections of these kidneys revealed the presence of largegrafts of human cells under the kidney capsule. Immunofluorescencestaining of these grafts showed that both SC-β cells and human isletgrafts contained high levels of insulin expressing cells (FIG. 12B).Those INS+ cells also co-expressed the canonical β cell transcriptionfactor PDX1 as expected for functional β cells. Analysis of insulin andglucagon staining further revealed that the SC-β cells remainedmonohormonal after transplantation (FIGS. 13A-13B). A minor populationof GCG+ a cells are also generated in this protocol as observed byimmunofluorescence and flow cytometry analyses (FIG. 7 and FIG. 10) andthose cells are also observed in vivo post-transplantation (FIG. 13).

It was further observed that insulin secretion in vitro increased overtime when media, particularly supplemented CMRL media containing Alk5inhibitor and T3 hormone, was used in the last differentiation step.Thus SC-β cells were cultured in vitro for an additional week (two weeksinstead of one week in this last step media) and were observed to seewhether they would also secrete more insulin in vivo. Aftertransplanting these aged cells, ten fold higher levels of insulin wereunexpectedly observed than the same number of transplanted younger SC-βcells (FIG. 12D). Thus the levels of insulin function achievable in vivowith human SC-β cells was similar to that achieved by transplantation ofhuman islets. Taken together these transplantation data suggest thatSC-β cells can provide an alternative clinical option for celltransplantation that does not rely on variable and limited supplies ofdonated cadaveric islets or on a poorly understood and poorlycontrollable in vivo maturation period from transplanted stem cellderived pancreatic progenitors.

Culture Media

To induce the in vitro maturation of at least some of the Pdx1-positive,NKX6-1-positive, insulin-positive endocrine cells into SC-β cells, thePdx1-positive, NKX6-1-positive, insulin-positive endocrine cells aremaintained in a suitable culture medium for a sufficient period of time.In the present invention, as mentioned above, it was found that usingCMRL media containing Alk5 inhibitor and T3 hormone allowed SC-β cellscultured in vitro to secrete more insulin in vivo. Adding additionalfactors to the CMRL media at the last step of differentiation (Stage 6)(FIG. 14A), however, was also found to generate a better glucosestimulated insulin secretion (GSIS) response by SC-β cells as measuredby either stimulation index between high and low glucose challenges orby the amount of insulin released. Such additional factors may include,but are not limited to, Sant1, XXI, and SSP. The stimulation index, ascalculated by the ratio of insulin secreted in high glucose (15 mM) tolow glucose (2.5 mM), was greater for the SC-β cells maintained in aCMRL media containing Alk5 inhibitor, T3 hormone and either Sant1, XXIor SSP than those SC-β cells maintained in only CMRL media, CMRL mediacontaining Alk5 inhibitor and T3 hormone, or S3 media containing Alk5inhibitor and T3 hormone (FIG. 14B). Furthermore, the amount of insulinsecreted was greater for the SC-β cells maintained in a CMRL mediacontaining Alk5 inhibitor, T3 hormone and either Sant1, XXI or SSP thanthose SC-β cells maintained in only CMRL media, CMRL media containingAlk5 inhibitor and T3 hormone, or S3 media containing Alk5 inhibitor andT3 hormone (FIG. 14C). Taken together, these data suggest that not onlyis maintaining SC-β cells in CMRL media important in the finaldifferentiation step to induce maturation of at least some of thePdx1-positive, NKX6-1-positive, insulin-positive endocrine cells intoSC-β cells, but the further addition of certain factors such as Sant1,XXI, or SSP to the CMRL media will enhance glucose stimulated insulinsecretion (GSIS) of the cells.

Enhancing Survival and Function of Cells

One challenge for the stem cell field has been generating a sufficientquantity of SC-β cells that can be useful for drug screening, diseasemodeling, or therapeutic use. For example, hES cells are technicallydifficult to culture, are vulnerable to apoptosis upon cellulardetachment and dissociation, undergo massive cell death particularlyafter complete dissociation, and have low cloning efficiency. (Watanabe,K. et al., Nature Biotechnology 25, 681-686 (2007)). As a result, thequantity of viable SC-β cells yielded may be low. By modify the protocolbetween Steps 3 and 6 in the manner shown in FIG. 15A, more pure NKX6.1+endocrine clusters can be generated (FIG. 15B) leading to more SC-βcells that can be therapeutically useful.

In Steps 3-5, for instance, cell survival can be improved by adding arho-associated protein kinase inhibitor, or a Rock inhibitor. It isbelieved that the addition of a Rock inhibitor enhances survival of EScells by preventing dissociation-induced apoptosis thus increasing theircloning efficiency. (Watanabe et al.) Examples of Rock inhibitorsinclude, but are not limited to, Y-27632, Fasudil/HA1077, and H-1152. Inthe present invention, treating the cells with a Rock inhibitor has beenshown to improve cell survival (FIG. 15C).

In addition to treating the cells with a Rocki inhibitor, the cells inSteps 3-4 can be treated with ActivinA alone or in combination withNicotinamide. Activin A and Nicotinamide have both been shown to improvecell survival. One way in which they improve cell survival is bydown-regulating SOX2, a marker for a pluripotent stem cell, from about81% to about 47% (FIG. 15D). Since SOX2 and NKX6-1 are mutuallyexclusive (FIG. 15E), down-regulation of SOX2 results in theup-regulation of NKX6-1, a marker for a mature pancreatic cell.

In Steps 5-6, cell survival can further be enhanced by treating cellswith staurosporine. Staurosporine is known to be an activator ofneuronal, glial, and stem cell-like neurosphere differentiation and isalso thought to induce apoptosis. (Schumacher, et al., Mol. Cell.Neurosci. 2003; 23(4): 669-680). In the present invention, the additionof staurosporine resulted in a near pure endocrine population byincreasing the percentage of ChromograninA, a pancreatic endocrine cellmarker, (FIG. 15F) and increasing the percentage of NKX6-1.C-peptide+cells (FIG. 15G).

Finally, cell survival can further be enhanced by treating cells with aculture medium containing Alk5i, T3 in combination with XXI, aγ-secretase inhibitor that improves β cell maturation. As shown in FIGS.15H and 15I, the addition of XXI to the medium at Steps 5 and 6 canincrease the NeuroD1+ population.

Taken together, these data suggest that adding certain compounds to thecells at various stages throughout the process is important in improvingcell survival and also enriching the population of cells that candifferentiate into therapeutically useful SC-β cells.

Utility of SC-β Cells for Translational Biology

A major challenge for the stem cell field has been generatingdifferentiated cell types that are close enough to their normallydeveloped counterparts to be useful for drug screening, diseasemodeling, or therapeutic use. SC-β cells generated using the newdifferentiation protocol as discussed herein appear to function both invitro and in vivo in a similar manner to primary human β cells. It wastherefore hypothesized that these cells could be useful fortranslational medicine (FIG. 16A).

One of the most common therapeutic strategies for treating Type 2diabetes is the administration of oral anti-diabetic medications. Manyof these pharmaceutical agents act directly on the β cell to increaseinsulin secretion. For example, sulfonylurea drugs increase secretion ofendogenous insulin through blocking potassium channels (Modi, 2007).Current drug screening on β cells is restricted to rodent islets,transformed cell lines or highly variable and limited supplies ofcadaveric human islets. Given that rodent metabolism only partiallymimics human metabolism, a reliable, consistent supply of human β cellsfor analysis would be very valuable. Here the inventors examined whetherSC-β cells could be used for therapeutic screening in vitro.

The inventors first tested whether SC-β cells could respond to existinganti-diabetic drugs from several different classes (FIG. 16B). LY2608204is glucokinase activator in clinical trials while liraglutide,tolbutamide, and nateglinide are examples of GLP-1 agonists andsecretagogues (sulfonylureas and meglitinides), respectively, that havebeen used clinically (Matschinsky, 2009; Modi, 2007; Vetere et al.,2014). Indeed SC-β cells showed a trend toward responding to treatmentwith these drugs by increasing insulin secretion in vitro following aglucose challenge (FIG. 16C). These data provide initial proof ofconcept that SC-β cells could provide a novel platform for future drugscreening.

Next the inventors tested whether SC-β cells could model β cellreplication. Increasing a patient's β cell mass may improve glycemiccontrol by producing more insulin in total without needing to increasethe amount of insulin produced per β cell. To date, few if any drugsthat have promoted β cell replication in rodent or cell line models havebeen translated to human β cells. Hormonal control of β cell replicationhas been suggested through studies of islet mass in human pregnancy androdent studies using prolactin treatment (Parsons et al., 1992; Labriolaet al., 2007; Brelje et al., 2004). The inventors tested whethertreatment with prolactin could increase β cell replication in a SC-βcell population. After culturing SC-β cells with prolactin, cells werestained and fixed for C-peptide and the proliferation marker Ki67 (FIGS.16D and 16E). Like primary human β cells, the baseline number of Ki67+proliferating SC-β cells was very low (0.20±0.08% Ki67+/C-peptide+)(FIG. 16F). Quantification of untreated and prolactin-treated cellsrevealed a trend toward increased proliferation downstream of prolactin(0.39±0.08% Ki67+/C-peptide+), suggesting that SC-β cells may be able torespond to proliferation cues in replication screens (FIG. 16F).

Finally, the inventors examined whether SC-β cells could be directlyuseful as a cell therapy for treating diabetes. Unlike type 2 diabetes,increasing β cell function or β cell replication via new pharmaceuticalsis likely to have little therapeutic benefit to patients with Type 1diabetes where the pancreatic β cells are destroyed by autoimmuneattack. These patients can be effectively treated by replacing lost βcells with donor allogeneic islets through the Edmonton transplantationprotocol (Shapiro et al., 2006).

One useful model of this kind of severe diabetes is the Akita mouse.Akita mice harbor a mutation in the insulin gene that leads to proteinmisfolding, complete and irreversible β cell failure, and progressivelymore severe hyperglycemia. Akita mice can be restored to normoglycemiavia mouse or human islet transplantation into the kidney capsule(Pearson et al., 2008). Therefore SC-β cells generated according to themethods described herein were tested to see if they could also functionto control diabetic hyperglycemia. The results showed thattransplantation of SC-β cells, but not PH INS+ cells of previousprotocols, into young Akita mice rapidly reversed the progressivelyworsening hyperglycemia observed in these animals (FIG. 16G). Fastingblood glucose measurements of mice transplanted with SC-β cells were onaverage less than 200 mg/dl whereas those transplanted with control PHcells showed progressively higher blood glucose levels above 400 mg/dl,as has been observed for non-transplanted Akita mice (FIG. 16G) (Pearsonet al., 2008). As expected, human insulin levels were high in SC-β celltransplanted animals and barely detectable in PH cell transplantedcontrol animals (FIG. 16H). The mice transplanted with SC-β cells alsoshowed better weight maintenance than control mice, indicative ofnormalized insulin function (FIG. 17). Thus SC-β cells are capablesecreting insulin and halting and reversing progressive hyperglycemia ina diabetic mouse model almost immediately following transplantation.

Discussion

The work described herein demonstrates that functional human β cells canbe directly generated from hPSC and hiPSC in vitro. Collectively, thedata described herein demonstrate that these cells (i.e., SC-β cells)function similarly to primary human β cells both in vitro and in vivopost-transplantation. SC-β cells generated according to the methodsdescribed herein present several new opportunities for β cell study andtherapy. Limited supplies of donated cadaveric islets, high variabilitybetween samples due to patient characteristics or isolation and thetrivially small amount of human β cell replication in vitro has severelylimited human β cell supply to-date. This limitation has restrictedtransplantation options for patients as well as high throughput drugscreening and disease modeling. A single 68 kg (1501b) patient requiresroughly 340-748 million transplanted islet cells to effectively resolvetype 1 diabetes via islet transplantation (McCall and Shapiro, 2012).The strategy described herein is both efficient and highly scalablemaking it practical for a single laboratory to grow hundreds of millionsto billions of SC-β cells at a time. A major clinical advantage of SC-βcells compared to previously described stem cell derived therapies fordiabetes is that these cells provide the first opportunity for a celltherapy that does not require a potentially unpredictable “black box”period of further differentiation in vivo.

Unlike primary human β cells, SC-β cells can also be generated fromcells of any desired genetic background. iPS cells from patients withdiabetes or other metabolic syndromes can now be derived anddifferentiated into SC-β cells for disease modeling and study of β cellfunction or susceptibility to stress or immune attack. Technologies likeTALEN and CRISPR enable genome editing of ES or iPS cells to incorporateand test variants identified by genetic analyses like genome wideassociation studies (GWAS). Similarly, β cell drug responses could nowbe compared between genetically matched pairs of mutant and non-mutant βcells (Ding et al., 2013). Identification and testing of novelbiomarkers for β cell function or pharmacogenetics is also enabled bythe combination of these technologies. Thus SC-β cells provide a novel,human-specific platform for in vitro drug discovery and characterizationin metabolism and diabetes.

The generation of SC-β cells also represents a potentially useful steptowards the future generation of islets and pancreatic organs.Incorporating pancreatic niche cells, like mesenchymal or endothelialcells into cultures of stem cell derived pancreatic cells may bebeneficial (Sneddon et al., 2012; Lammert et al., 2001). Other evidencesuggests that the presence of alpha and delta cells may be important forprecise tuning of normal cell function (Rodriguez-Diaz et al., 2011).Furthermore, tissue engineering of a pancreatic organ will requireincorporation of functional exocrine and ductal tissue, potentially incarefully specified architecture. The generation of SC-β cellsrepresents a step forward towards making a clinical impact throughharnessing stem cell biology.

Experimental Procedures

Cell Culture

hPSC lines were maintained undifferentiated in mTESR1 (StemCellTechnologies Inc.; 05850) in 500 ml stir flasks (Corning, VWR;89089-814) placed on a 9-Position stir plate set at rotation rate of 70rpm in a 37° C. incubator, 5% C02, and 100% humidity. The line HUES8 wasthe primary line utilized. Cells were dispersed with Accutase and wereseeded as single cells at 0.5 milion/ml in mTESR with 10 μM Y27632(Abcam; ab120129). mTESR1 media was changed (w/o Y27632) 48 hours later.Cultures were split 24 hours after that media change to keep clusterdiameter size <300 μm. Cultures were regularly tested for pathogens,karyotype and for maintenance of pluripotency markers.

Exemplary media used for directed differentiation were as follows:

S1 media: MCDB131 (Cellgro; 15-100-CV)+8 mM D-(+)-Glucose (Sigma;G7528)+2.46 g/L NaHCO₃(Sigma; S3817)+2% FAF-BSA (Proliant; 68700)+ITS-X(Invitrogen; 51500056) 1:50.000+2 mM GlutaMAX™ (Invitrogen;35050079)+0.25 mM Vitamin C (Sigma Aldrich; A4544)+1% Pen/Strep(Cellgro; 30-002-CI).

S2 media: MCDB131+8 mM D-Glucose+1.23 g/L NaHCO₃+2% FAF-BSA+ITS-X1:50.000+2 mM1 GlutaMAX™+0.25 mM Vitamin C+1% Pen/Strep.

S3 media: MCDB131+8 mM D-Glucose+1.23 g/L NaHCO₃+2% FAF-BSA+ITS-X1:200+2 mM1 GlutaMAX™+0.25 mM Vitamin C+1% Pen/Strep

BE5 media: MCDB131+20 mM D-Glucose+1.754 g/L NaHCO₃+2% FAF-BSA+ITS-X1:200+2 mM GlutaMAX™+0.25 mM Vitamin C+1% Pen/Strep+Heparin 10 μg/ml(Sigma; H3149).

CMRLS: CMRL 1066 Supplemented (Mediatech; 99-603-CV)+10% FBS (HyClone™,VWR; 16777)+1% Pen/Strep.

All media were filter sterilized through a 0.22 μm bottle top filter(Corning). For all following media changes, small molecules and growthfactors were added to the base media immediately before media change ina low-light hood.

For initiation of SC-β cell differentiation, cells were seeded at 0.5million/ml and differentiation was started 48 hours later. Media changeswere as follows—

Day 1: S+100 ng/ml Activin A (R&D Systems; 338-AC)+3 μM Chir99021(Stemgent; 04-0004-10)).

Day 2: S+100 ng/ml Activin A. Days 4, 6: S2+50 ng/ml KGF (Peprotech;AF-100-19)).

Days 7, 8: S3+50 ng/ml KGF+0.25 μM Sant1 (Sigma; S4572)+2 μM Retinoicacid (RA) (Sigma; R2625)+200 nM LDN193189 (only Day 7) (Sigma;SML0559)+500 nM PdBU (EMD Millipore; 524390)).

Days 9, 11, 13: S3+50 ng/ml KGF+0.25 μM Sant1+100 nM RA.

Days 14, 16: BE5+0.25 μM Sant1+50 nM RA+1 μM XXI (EMD Millipore;565790)+10 μM Alk5i II (Axxora; ALX-270-445)+1 μML-3,3′,5-Triiodothyronine (T3) (EMD Millipore; 64245)+20 ng/mlBetacellulin (Thermo Fisher Scientific; 50932345)).

Days 18, 20: BE5+25 nM RA+1 μM XXI+10 μM Alk5i II+1 μM T3+20 ng/mlBetacellulin.

Days 21-35 (media change every second day): CMRLS+10 μM Alk5i II+1 μMT3. Cells were tested by in vitro assays after between days 28 and 32.Cells were transplanted on day 28, unless otherwise noted.

Alternatively, Days 21-35: CMRLS+10 μM Alk5i II+1 μM T3+Sant1.

Alternatively, Days 21-35: CMRLS+10 μM Alk5i II+1 μM T3+XXI.

Alternatively, Days 21-35: CMRLS+10 μM Alk5i II+1 μM T3+SSP.

For generation of PH protocol cells to mimic previous publications, thesame differentiation protocol was used until day 14. On days 14 and 16,cells were fed with S3+1 μM Alk5i II, 200 nM LDN193189, and 100 nM RA(Rezania et al., 2012). On days 18 and onward, cells were fed everyother day with S3+100 nM RA. Cells were maintained in this media untilexperimental analysis. Cells were maintained in culture and tested afterthe same number of days in differentiation media as the SC-β cells tocontrol for the impact of time.

Flow Cytometry

Cells were dispersed into single-cell suspension by incubation in TrypLEExpress at 37° C. for 10-30 min in a 15 ml falcon tube. Starting atstage 5, clusters take longer to dissociate into single cells. Clustersshould be incubated with TrypLE Express until they dissociate to singlecells upon mixing by pipetting gently up and down with a P1000. TheTrypLE Express was quenched with 3-4 times media and cells were spundown for 5 min at 1000 rpm. Cells were washed twice in PBS andtransferred to a 1.7 ml Safe seal microcentrifuge tube (Bioscience Inc.;11510). Make sure having 1-10 mio cells/tube and use 0.5-1 ml volumes inthe following. Cells were resuspended in 4% paraformaldehyde (PFA)(Electron Microscopy Science Nm; 15710) and incubated on ice for 30 min.Cells were then washed 3 times in PBS followed by incubation in blockingbuffer (PBS+0.1% TritonX100 (VWR; EM-9400)+5% donkey serum (JacksonImmunoresearch; 017-000-121)) on ice for 1 hour. After fixation cellsare more resistant to centrifugation, so after fixation allcentrifugations were done at 3000 g for 3 min. Cells were thenresuspended in blocking buffer with primary antibodies and incubated at4° C. overnight.

Primary antibodies were used 1:300 unless otherwise noted: Goatanti-human PDX-1/IPF1 (R&D Systems; AF2419), mouse anti-NKX6-1(University of Iowa, Developmental Hybridoma Bank; F55A12-supernatant)(1:100), rabbit anti-Chromogranin A (Abcam; ab15160), rat anti-insulin(pro-)/C-peptide (Developmental Studies Hybridoma Bank at the Universityof Iowa; GN-ID4) (need to add glucagon and somatostatin). Cells werewashed 2 times in blocking buffer and then incubated in blocking bufferwith secondary antibodies on ice for 2 hours (protected from light).Secondary antibodies conjugated to Alexa Fluor 488, 647 (LifeTechnologies) or PE (Thermo Fisher Scientific; NC9774252) were used tovisualize primary antibodies. Cells were then washed 3 times in sortingbuffer (PBS+0.5% BSA (Sigma; A8412)) and finally resuspended in 500-700μl sorting buffer, filtered through a 40 μm nylon mash into FACS tubes(BD Falcon; 352235), and analyzed using the LSR-II FACS machine (BDBiosciences) with at least 30,000 events being recorded. Analysis of theresults was done using FlowJo software.

Immunofluorescence

Cells were dispersed and plated onto 96 well plates. After one day ofculture, cells were washed in PBS and fixed in 4% PFA for 20 min at RT.Following 3 washes with PBS, cells were blocked for 1 hour at RT inPBS+0.1% Triton X-100+5% donkey serum. All primary antibody incubationswere done overnight at 4° C. in blocking solution at a 1:500 dilution:Goat anti-human PDX-1/IPF1, mouse anti-NKX6-1, rabbit anti-ChromograninA, rat anti-insulin (pro-)/C-peptide, Ki67. Cells were washed 2 times inPBS the next day, followed by secondary antibody incubation for 1 hourat RT at a 1:500 dilution (protected from light). Secondary antibodiesconjugated to Alexa Fluor 488, 594 or 647 (Life Technologies) were usedto visualize primary antibodies. Following 3 washes with PBS, all nucleiwere visualized by staining with DAPI (Invitrogen; D1306).Representative images were taken using an Olympus IX51 Microscope orZeiss LSC 700 confocal microscope. The percentage of target cell-typeswas quantified using the Array Scan Cellomics high-content screeningsystem. Here 30 images were acquired per well and quantified. Cellslabeled by antibody staining and total cell number (based on DAPI nucleistaining) were quantified to obtain percentages of target cell types.

Immunohistochemistry

Cell clusters or islets were fixed by immersion in 4% PFA for 1 hour atRT. Samples were then washed 3 times with PBS, embedded in Histogel™,and sectioned for histological analysis. 10 m sections were thensubjected to deparrafinization using Histoclear (Thermoscientific;C78-2-G) and rehydrated. For antigen retrieval slides were emerged in0.1M EDTA (Ambion; AM9261) and placed in a pressure cooker (Proteogenix;2100 Retriever) for two hours. Slides were then blocked with PBS+0.1%Triton X-100+5% donkey serum for 1 hour at RT, followed by incubation inblocking solution with primary antibodies overnight at 4° C. Thefollowing primary antibodies were used 1:100 unless otherwise noted:Goat anti-human PDX-1/IPF1, mouse anti-NKX6-1, rabbit anti-ChromograninA, rat anti-insulin (pro-)/C-peptide, glucagon, somatostatin. Cells werewashed 2 times in PBS the next day, followed by secondary antibodyincubation for 2 hour at RT (protected from light). Secondary antibodiesconjugated to Alexa Fluor 488 or 594 were used to visualize primaryantibodies. Following washes with PBS, the histology slides were mountedin Vectashield mounting medium with DAPI (Vector Laboratories; H-1200),covered with coverslips and sealed with nail polish. Representativeimages were taken using an Olympus IX51 Microscope or Zeiss LSM 510 or710 confocal microscope.

Glucose Stimulated Insulin Secretion

Krebs buffer (Krb): Di H₂O with 128 mM NaCl, 5 mM KCl, 2.7 mM CaCl₂),1.2 mM MgCl₂, 1 mM Na₂HPO₄, 1.2 mM KH₂PO₄, 5 mM NaHCO₃, 10 mM HEPES(Life Technologies; Ser. No. 15/630,080), 0.1% BSA (Proliant; 68700).Solutions were equilibrated to 37° C. in incubator and 1 ml volumes wereused in the following protocol. Approximately 0.5 million cells asclusters were transferred to autoclaved 1.7 ml Safe seal microcentrifugetubes and washed 2 times in Krb. Clusters were then pre-incubated in Krbwith 2 mM glucose for 2 hours in incubator to remove residual insulin.It is worth noting that for all incubations tube lids were left open andcovered by a lid that allowed for air exchange. Clusters were washed 2times in Krb and then incubated in Krb containing 2 mM Glucose forexactly 30 min and 200 ul of the supernatant collected after incubation(low glucose sample). Clusters were washed 2 times in Krb and thenincubated in Krb with 20 mM Glucose for exactly 30 min and 200 ul ofsupernatant was collected after incubation (high glucose sample).Challenging with low and high glucose was repeated two additional times(3 paired challenges in total). Finally, clusters were washed 2 times inKrb and then incubated in Krb with 2 mM Glucose+30 mM KCl for exactly 30min and 200 ul of supernatant was collected after incubation (KClpolarization challenge sample). After the KCl challenge, clusters weredispersed using Tryple and counted by Viacell (manufacturer) tonormalize insulin secretion amounts by cell number.

Supernatant samples containing secreted insulin were then processedusing the Human Ultrasensitive Insulin ELISA (ALPCO Diagnostics;80-INSHUU-E01.1) and samples were measured by a FLUOstar optimaspectrophotometer (BMG lantech) at 450 nm. If the ELISA was notperformed on the same day, samples were stored at −80° C. In order toget insulin concentrations within the range of the ELISA, diluting thesamples between 1:100 and 1:200 in PBS was usually sufficient.

Electron Microscopy

Cell clusters were fixed with a mixture containing 1.25% PFA, 2.5%glutaraldehyde, and 0.03% picric acid in 0.1 M sodium cocodylate buffer(pH 7.4) for at least 2 hours at RT. Cell clusters were then washed in0.1M cacodylate buffer and post-fixed with a mixture of 1% Osmiumtetroxide (OsO4) and 1.5% Potassium ferrocyanide (KFeCN6) for at least 2hours at RT, washed in 0.1M cacodylate buffer and post-fixed with 1%Osmiumtetroxide (OsO4)/1.5% Potassiumferrocyanide (KFeCN6) for 1 hour,washed in water 3× and stained in 1% aqueous uranyl acetate for 1 hourfollowed by 2 washes in water and subsequent dehydration in grades ofalcohol (10 min each; 50%, 70%, 90%, 2×10 min 100%). The samples werethen incubated in propyleneoxide for 1 hour and infiltrated ON in a 1:1mixture of propyleneoxide and TAAB Epon (Marivac Canada Inc. St.Laurent, Canada). The following day the samples were embedded in TAABEpon and polymerized at 60° C. for 48 hours. Ultrathin sections (about60 nm) were cut on a Reichert Ultracut-S microtome, picked up on tocopper grids, stained with 0.2% lead citrate and examined in a JEOL1200EX Transmission electron microscope or a TecnaiG² Spirit BioTWIN.Images were recorded with an AMT 2 k CCD camera and analyzed usingImageJ software.

SCID-Beige Transplantation Studies

5 million hPSC derived cells as clusters were resuspended in RPMI1640media (Life technologies; 11875-093), aliquoted into PCR tube with thevolume of 200 uL, and kept on ice for 5 to 10 minutes before the loadinginto the catheter. For the preparation of cell loading, each catheter,infusion set 23G×¾″ (Terumo; SB*S23BL) connected to 1 mL syringe, wasrinsed with 1 ml of 5% FBS serum (Corning; 35-011-CV) added RPMI mediathen loaded with 0.6 ml of no serum added RPMI media. Clusters wereloaded through the tip of the catheter needle and placed vertically tosettle on the bottom of the catheter tubing and near the top of theneedle by gravity for 5 minutes in room temperature. During the cellpreparation step, immunodeficient (SCID/Beige) mice (what age?) wereanesthetized with avertin 1.25% (250 mg/kg; 0.5 ml/25 g 1.25%Avertin/body weight), and left ventricle surgical site was shaved anddisinfected with betadine and alcohol. Incision of about 1 cm was madeto expose the kidney and clusters were injected under the kidney capsuleby inserting catheter needle under the kidney capsule and injectingabout 100 ul volume containing 5 million equivalent cells of clusters.Abdominal cavity was closed with PDS absorbable sutures (POLY-DOX;2016-06)) and skin with surgical clips (Kent Scientific Corp;INS750346-2). Mice were placed on a micro-temp circulating pump andblanket (˜37° C.) during the surgery/recovery period to aid in the rapidrecovery of mice following anesthesia and given a dose of 5 mg/mkgcarprofen right after the surgery and 24 hours after the initial dose.The average time for transplantation is approximately 3 minutes permouse. Wound clips were removed 14 days after surgery and the mice weremonitored twice a week.

After two weeks of recovery from surgery, the presence of human insulinand the glucose-responsiveness of the transplanted cells were assessedby per forming glucose tolerance test (GTT). After fasting the mice for16 hours overnight, GTT was performed by IP injection of 2 gD-(+)-glucose/1 kg body weight and blood collection of pre and/or postinjection through facial vein puncture using lancet (Feather; 2017-01).Human insulin was subsequently quantified using the human insulin ELISAkit (ALPCO Diagnostics; 80-INSHUU-E01.1). Grafts were dissected from theSCID mice, fixed in 4% PFA (Electron Microscopy Science Nm; 15710),embedded in paraffin, and sectioned for histological analysis.

Calcium Imaging

About 10 to 20 hPSC derived clusters were plated on 96 well plate coatedwith MATRIGEL™ and allowed to adhere for 24 hours in a 37° C. incubator,5% CO₂, and 100% humidity. Clusters were washed with prewarmed (37° C. )Krebs buffer added with 2.5 mM glucose then incubated with 50 μMCa²⁺-sensitive fluorescent probe Fluo4-AM (Life Technologies; F14217) in2.5 mM glucose Krb buffer for 45 minutes in 37° C. incubator. Clusterswere washed with 2.5 mM glucose Krb buffer then incubated further in 37°C. incubator for additional 15 minutes. Clusters were then right awaystaged in the AxioZoom V16 microscope (Carl Zeiss) for acquirement ofhigh resolution time series imaging of multiple batches of clusters indifferent wells. Fluo-4 was excited at 488 nm and its emission wasrecorded between 490 and 560 nm. Time series images were acquired atsingle cell resolution of 80× magnification in every 17 seconds and upto 10 wells were imaged in one imaging. Progression of glucosechallenges and time of the stimulation during imaging was as follows.Imaging started with 5 minute stimulation of 2 mM glucose in Krbfollowed by 5 minute stimulation of 20 mM glucose in Krb buffer. Theselow then high glucose stimulations were repeated two more times, thenimaging ended with 5 minute stimulation of 30 mM KCl in Krb buffer andwith the total imaging time of 35 minutes. Between the stimulations,imaging was stopped and clusters were washed with 2 mM glucose Krbbuffer and then added with the next glucose Krb buffer. Fluorescenceintensity changes during 35 minutes of imaging were analyzed in thesingle cell resolution using Imagej/Fiji by applying StackReg to correctfor the movement of the clusters over the course of the imaging, and ROImanager to measure the fluorescence intensity of the cells throughoutthe imaging. All 7 stimulations of 5 minute clips were made into onemovie using VirtualDub and published in youtube for data sharing.

Intracellular Flow Cytometry and Gene Expression Analysis

MARIS was carried out as described in Hrvatin et al., 2014. Cells wereharvested in single cell suspension, fixed in 4% PFA on ice, incubatedwith primary antibody and then secondary antibodies in buffer containingRNasin. Cells were then sorted by FACS to obtain at least 100,000 cellsper sample. Samples were subsequently incubated in digestion Buffer at50° C. for 3 hours, prior to RNA isolation. RNA concentration wasquantified using Nanodrop 1000. Double-stranded cDNA was generated byreverse transcription from at least 100 ng of total RNA. Amplified mRNA(cRNA) was then generated by In vitro transcription overnight withbiotin-labeled nucleotides and concentrated by vacuum centrifugation at30° C. At least 750 ng cRNA per sample was hybridized to Human HT-12Expression BeadChips (Illumina) using the Whole-Genome Expression DirectHybridization kit (Illumina). Chips were scanned on the IlluminaBeadstation 500. Raw data was adjusted by background subtraction andrank-invariant normalization. Before calculating fold change, an offsetof 20 was added to all probe set means to eliminate negative signals.The p-values for differences between mean signals were calculated inGenomeStudio by t-test and corrected for multiple hypotheses testing bythe Benjamini-Hochberg method in combination with the Illumina customfalse discovery rate (FDR) model.

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1. An in vitro composition that comprises a TGF-beta superfamily growthfactor, a sonic hedgehog pathway inhibitor, and a plurality ofnon-native human pancreatic progenitor cells that express PDX1.
 2. Thecomposition of claim 1, wherein the plurality of non-native pancreaticprogenitor cells that express PDX1 comprise cells that express PDX1 butnot NKX6.1.
 3. The composition of claim 2, wherein the plurality ofnon-native pancreatic progenitor cells that express PDX1 furthercomprise cells that express both PDX1 and NKX6.1.
 4. The composition ofclaim 1, wherein the TGF-beta superfamily growth factor is selected fromthe group consisting of: Activin A, growth differentiation factor 8(GDF8), growth differentiation factor 11 (GDF11), IDE1 and IDE2.
 5. Thecomposition of claim 1, wherein the TGF-beta superfamily growth factorcomprises Activin A.
 6. The composition of claim 1, wherein the TGF-betasuperfamily growth factor comprises GDF8 or GDF11.
 7. The composition ofclaim 1, wherein the TGF-beta superfamily growth factor has aconcentration of 10 ng/mL-1000 ng/mL.
 8. The composition of claim 1,wherein the composition further comprises one or more of a FGF familygrowth factor, and a retinoic acid signaling pathway activator. 9.(canceled)
 10. The composition of claim 8, wherein the compositioncomprises the FGF family growth factor and the retinoic acid signalingpathway activator.
 11. The composition of claim 8, wherein thecomposition comprises the FGF family growth factor, and wherein the FGFfamily growth factor is selected from the group consisting of: KGF,FGF2, FGF8B, FGF10, and FGF21.
 12. The composition of claim 1, whereinthe sonic hedgehog pathway inhibitor is selected from the groupconsisting of: Sant1, Sant2, Sant3, Sant4, Cur61414, forskolin,tomatidine, AY9944, and triparanol.
 13. The composition of claim 8,wherein the composition comprises the retinoic acid signaling pathwayactivator, and wherein the retinoic acid signaling pathway activator isselected from the group consisting of: retinoic acid; CD 1530; AM 580;TTNPB; CD 437; Ch 55; BMS 961; AC 261066; AC 55649; AM 80; BMS 753;tazarotene; adapalene; CD 2314; DEAB; BMS 195614; ER 50891; BMS 493; CD2665; LE 135; BMS 453; and MM
 11253. 14. The composition of claim 10,wherein: a) the TGF-beta superfamily growth factor is selected from thegroup consisting of: Activin A, growth differentiation factor 8 (GDF8),growth differentiation factor 11 (GDF11), IDE1 and IDE2; b) the FGFfamily growth factor is selected from the group consisting of: KGF,FGF2, FGF8B, FGF10, and FGF21; c) the sonic hedgehog pathway inhibitoris selected from the group consisting of: Sant1, Sant2, Sant3, Sant4,Cur61414, forskolin, tomatidine, AY9944, and triparanol; and d) theretinoic acid signaling pathway activator is selected from the groupconsisting of: retinoic acid, CD 1530; AM 580; TTNPB; CD 437; Ch 55; BMS961; AC 261066; AC 55649; AM 80; BMS 753; tazarotene; adapalene; CD2314; DEAB; BMS 195614; ER 50891; BMS 493; CD 2665; LE 135; BMS 453; andMM
 11253. 15. The composition of claim 10, wherein the TGF-betasuperfamily growth factor comprises Activin A, the FGF family growthfactor comprises KGF, the sonic hedgehog pathway inhibitor comprisesSANT1, and the retinoic acid signaling pathway activator comprisesretinoic acid.
 16. The composition of claim 1, wherein the non-nativepancreatic progenitor cells that express PDX1 are genetically modifiedcells or progenies thereof.
 17. A method of in vitro celldifferentiation, comprising: contacting an in vitro cell populationcomprising pancreatic progenitor cells that express PDX1 but not NKX6.1with a TGF-beta superfamily growth factor, an FGF family growth factor,a sonic hedgehog pathway inhibitor, and a retinoic acid signalingpathway activator, wherein the method results in a plurality of thepancreatic progenitor cells expressing PDX1 but not NKX6.1 in thepopulation to differentiate into pancreatic progenitor cells thatexpress PDX1 and NKX6.1.
 18. The method of claim 17, wherein the cellpopulation is contacted with the TGF-beta superfamily growth factor, theFGF family growth factor, the sonic hedgehog pathway inhibitor, and theretinoic acid signaling pathway activator for 4, 6, or 8 days.
 19. Themethod of claim 17, wherein the TGF-beta superfamily growth factor isselected from the group consisting of: Activin A, growth differentiationfactor 8 (GDF8), growth differentiation factor 11 (GDF11), IDE1 andIDE2.
 20. The method of claim 18, wherein the TGF-beta superfamilygrowth factor comprises Activin A.
 21. The method of claim 18, whereinthe TGF-beta superfamily growth factor comprises GDF8 or GDF11.
 22. Themethod of claim 17, wherein the FGF family growth factor is selectedfrom the group consisting of: KGF, FGF2, FGF8B, FGF10, and FGF21. 23.The method of claim 17, wherein the sonic hedgehog pathway inhibitor isselected from the group consisting of: Sant1, Sant2, Sant3, Sant4,Cur61414, forskolin, tomatidine, AY9944, and triparanol.
 24. The methodof claim 17, wherein the retinoic acid signaling pathway activator isselected from the group consisting of: retinoic acid; CD 1530; AM 580;TTNPB; CD 437; Ch 55; BMS 961; AC 261066; AC 55649; AM 80; BMS 753;tazarotene; adapalene; CD 2314; DEAB; BMS 195614; ER 50891; BMS 493; CD2665; LE 135; BMS 453; and MM
 11253. 25. The method of claim 17,wherein: a) the TGF-beta superfamily growth factor is selected from thegroup consisting of: Activin A, growth differentiation factor 8 (GDF8),growth differentiation factor 11 (GDF11), IDE1 and IDE2; b) the FGFfamily growth factor is selected from the group consisting of: KGF,FGF2, FGF8B, FGF10, and FGF21; c) the sonic hedgehog pathway inhibitoris selected from the group consisting of: Sant1, Sant2, Sant3, Sant4,Cur61414, forskolin, tomatidine, AY9944, and triparanol; and d) theretinoic acid signaling pathway activator is selected from the groupconsisting of: retinoic acid, CD 1530; AM 580; TTNPB; CD 437; Ch 55; BMS961; AC 261066; AC 55649; AM 80; BMS 753; tazarotene; adapalene; CD2314; DEAB; BMS 195614; ER 50891; BMS 493; CD 2665; LE 135; BMS 453; andMM
 11253. 26. The method of claim 17, wherein the TGF-beta superfamilygrowth factor has a concentration of 10 ng/mL-1000 ng/mL.
 27. The methodof claim 17, wherein the TGF-beta superfamily growth factor comprisesActivin A, the FGF family growth factor comprises KGF, the sonichedgehog pathway inhibitor comprises SANT1, and the retinoic acidsignaling pathway activator comprises retinoic acid.
 28. The method ofclaim 27, wherein the cell population is contacted with the TGF-betasuperfamily growth factor, the FGF family growth factor, the sonichedgehog pathway inhibitor, and the retinoic acid signaling pathwayactivator for 4, 6, or 8 days.